Technical Field
[0001] The present invention relates to a structure of a diffraction-grating type sensor
chip (surface plasmon resonance sensor chip) for analyzing a sample using surface
plasmon resonance (SPR), and specifically, to a structure of a sensor chip that is
suitable for use with small-sized clinical apparatus or an HPLC detector, and also
relates to a method of and an apparatus for analyzing a sample using the sensor chip.
Background Art
[0002] Currently, in areas such as biochemistry or medical inspection, an analysis method
using surface plasmon resonance (SPR) is one of the known methods of quantitatively
and/or qualitatively analyzing a sample fluid containing target species such as chemical
species, biochemical species, or biological species. The surface plasmon resonance
is the phenomenon that is excited by the resonance of a surface plasmon wave, which
is induced along the metal surface by light incident on a metal layer, and an evanescent
wave, which is generated by the incident light. The surface plasmon resonance is dependent
on the wavelength and the angle of the incident light: characteristically, when surface
plasmon resonance is excited, the optical component having a specific incident angle
or a specific wavelength transfers its optical energy to the surface plasmon wave,
and the reflected optical component that has the corresponding incident angle or wavelength
therefore decreases markedly.
[0003] In order to induce the surface plasmon resonance, it is necessary to use a metal
for making a specific surface plasmon wave and an optical structure for inducing an
evanescent wave that can be resonant with the surface plasmon wave. As the examples
of the optical structure for inducing an evanescent wave, two kinds of structures
are currently known: one is an optical structure utilizing total reflection with a
prism, and the other is an optical structure utilizing a diffraction grating. An element
that combines the metal with one of these optical structures is commonly called a
surface plasmon resonance sensor chip (hereinafter called simply a sensor chip).
[0004] A sensor chip is typically constructed by laminating a substrate with a metal layer
and by applying on the metal layer a binding substance (a ligand, a molecular-recognition
element), which can bind specifically to a specific target species (the subject of
analysis) through interaction, to thereby immobilize it. By making a sample in contact
with the surface of the metal layer, on which the binding substance is immobilized,
the target species in the sample is captured by the binding substance. The surface
plasmon resonance is also dependent on the refractive index of a medium present on
the surface of the metal layer: when the refractive index of the medium varies, the
resonance angle varies accordingly if the wavelength is constant, while the resonance
wavelength varies accordingly if the incident angle is constant. It is therefore possible
to analyze the refractive index of the medium present on the surface of the metal
layer by determining the resonance angle or the resonance wavelength based on the
intensity of the reflected light. In this case, the variation in the refractive index
of the medium present on the surface of the metal layer is correlated with the amount
of substance of the target species captured by the binding substance, namely, the
variation in the concentration of the target species relative to the sample. Consequently,
by determining the resonance angle or the resonance wavelength at which surface plasmon
resonance occurs, it is possible to analyze the concentration of the target species
in the sample or the like.
[0005] Of these two sensor chips, a prism type sensor chip is generally made up of a sensor-chip
body (a transparent substrate laminated with a metal layer) and a prism. A sensor
chip is basically thrown away after a single use. However, if not only the sensor-chip
body but also the prism, which is expensive, are made disposable, the cost of measurement
becomes very high. For this reason, this type of sensor chip is commonly made in such
a manner that the sensor-chip body and the prism can be separated. When the sensor
chip is used, the prism is brought into close contact with the sensor-chip body while
light is entered into the prism and the reflected light is detected, thereby a measurement
being carried out.
[0006] On the other hand, a diffraction-grating type sensor chip is constructed by laminating
a transparent substrate, whose surface has an uneven form having projections and depressions
(a grating), with a metal layer. Since the metal layer is layered over the uneven
form, the surface of the metal layer also takes an uneven form, which uneven form
on the surface of the metal layer functions as a diffraction grating. Using a high-order
diffraction light, an evanescent wave is generated along the conductor surface. When
the wave number and the angular frequency of the surface progressive wave agree with
those of the surface plasmon wave of the metal surface, a resonance occurs and causes
a decrease in the reflectance. It is thereby possible to detect the properties of
the metal surface or the properties of substances in contact with the metal surface
(refer to Japanese Patent Number 1903135, Japanese Patent Number 2502222, and others).
[0007] FIG. 51 is a dispersion relationship diagram showing the relations between the angular
frequencies ω and the wave numbers k of the surface plasmon wave, the evanescent wave
and the irradiated light in a diffraction-grating type sensor chip. In the figure,
curved lines A1, A2 both indicate the relation between the angular frequency ω and
the wave number k of the surface plasmon wave, curved line A1 indicating the case
in which the refractive index of the medium is higher than that in the case indicated
by curved line A2. Straight line B0 indicates the relation between the angular frequency
ω and the wave number k of the irradiated light, while straight line B1 indicates
the relation between the angular frequency ω and the wave number k of the evanescent
wave of a specific order corresponding to the irradiated light. In FIG. 51, each of
intersection points P11, P12 of curved lines A1, A2 and straight line B1 indicates
a resonance point at which surface plasmon resonance will occur, and the wavelength
and the incident angle corresponding to the resonance point are equal to the resonance
wavelength and the resonance angle. At the resonance point, the intensity of the reflected
light takes its minimum value.
[0008] Since a sensor chip of this type dose not use any expensive element such as a prism,
which is used for the prism type, it is so inexpensive that it can be made disposable.
Further, since a sensor chip of this type dose not need the task of making the prism
in close contact with the sensor-chip body, which task is necessary for the prism
type, it has another advantage in its high degree of reproducibility of measurement
values without being affected by defects such as variations in the degree of contact.
[0009] Besides, since the prism type sensor chip is constructed such that the incident light
and the reflected light pass through the prism, it has the limitations on the possible
diameter of a light beam and the area that can be irradiated with a light beam. On
the contrary, since the diffraction-grating type sensor chip does not have such limitations,
it is possible to use a light beam of large diameter, or is also to irradiate any
desired position with a light beam. The diffraction-grating type sensor chip therefore
has the advantages over the prism type in that it is possible to inspect a large area
at a time, and also in that it is possible to inspect any desired position on the
sensor chip. With such advantages as have been described, expectations are now rising
with respect to the diffraction-grating type sensor chip.
[0010] The common methods of analyzing a sample using a diffraction-grating type sensor
chip are: the analysis method in which the incident angle is held constant while the
wavelength is varied (or a multicomponent light is entered) and the resonance wavelength
is thereby detected; and the analysis method in which the incident wavelength is held
constant while the angle is varied and the resonance angle is thereby detected.
[0011] Of these methods, the former is commonly carried out by irradiating with a white
light (multicomponent light) at a constant incident angle and by measuring the reflectance
of the reflected light for each wavelength to thereby measure the peak of the absorption
by surface plasmon resonance. Because of the limitations of the measurement range,
the reflectance is usually measured for the reflected light within a specific range
(angular frequency ω1-ω2), as shown in FIG. 51.
[0012] However, the relation between the wave number and the frequency of the surface plasmon
wave is determined based on the dielectric constant (i.e. refractive index) of each
the metal layer and the sample (dielectric substance). Hence there is a possibility
that the resonance point goes out of the measurement range depending on the refractive
index value of the sample, as is the case of resonance point P12 shown in FIG. 51.
Especially in the case where different binding substances are immobilized on plural
spots so as to detect a sample having a wide distribution of dielectric constant (refractive
index), or in the case where some new substance (for example, pigmentation deposits
caused by enzyme reaction) is generated in stages during analysis and causes the change
in the dielectric constant (refractive index) of each of plural spot, there is a possibility
that if a measurement is carried out for these spots at a time, a resonance phenomenon
is detectable in some spots while undetectable in the other spots. In such cases,
in order to measure a resonance phenomenon for every spot, it becomes necessary to
make some readjustments to the optical system, for example, to modify the incident
angle.
[0013] It is also a possible idea of broadening the measurement range to widening a wavelength
band so that all the resonance points come within the measurement range. However,
if the wavelength band is too wide, although a broad range of wavelength can be detected,
there arises a decline in resolving power to become another problem.
[0014] Meanwhile, as the detection method of the resonance angle under the condition that
the incident wavelength is held constant, the following four methods are generally
known.
[0015] The first method is a method in which the incident angle of the incident light is
varied (this process is called angle scan) while the detection angle of a detector
for detecting the reflected light is varied in synchronism with the incident angle
to thereby detect the resonance angle.
[0016] The second method is a method in which the angle scan of the incident light is carried
out, as in the case of the first method angle, while the reflected light is detected
using a fixed type detector (an array type detector such as a CCD) to thereby detect
the resonance angle.
[0017] The third method is a method in which a wedge-shaped light is used as the incident
light while the detection angle of the detector is varied within the limits of the
reflection angle of the reflected light to thereby detect the resonance angle. More
specifically, as illustrated by FIG. 52(a), light of a single wavelength is emitted
from a light source 502 with a predetermined angle of divergence, and is entered into
the surface of the metal layer of a sensor chip 501 at a predetermined angle but perpendicularly.
Then the reflected light is relayed by a reflecting mirror 503 and detected by a light
detector (photo diode array) 504. In this case, as illustrated by FIG. 52(b), since
the incident angle of the entered light varies according to the incident position
on the sensor chip 501, it is possible to obtain the same results as are obtained
in the case where the single-wavelength light is used while the incident angle is
changed continuously from angle θa to angle θb.
[0018] The fourth method is a method in which a wedge-shaped light is used as the incident
light, as in the case of the third method, while the reflected light is detected by
an angle-fixed type detector to thereby detect the resonance angle.
[0019] In addition to the above methods, there is another known method in which analysis
is carried out under the condition that both the incident angle and the incident wavelength
are constant. The distribution of the intensity of the reflected light over the incident
angle or the incident wavelength changes according to the amount of target species
captured by the binding substance, namely, the concentration of target species in
the sample. In this method (the fifth method), the intensity of the reflected light
is measured under the condition that both the incident angle and the incident wavelength
are constant, and the analysis of the concentration and others is carried out based
on the variation of the intensity under the condition (variation compared with the
case where the sample is not in contact).
[0020] However, the above analysis methods each have the following problems. Regarding the
first and second methods, it is required to use a driving mechanism for changing the
angle of the light axis of the light source in order to carry out the angle scan of
the incident light, resulting in a cost increase. Since the requirement of a driving
mechanism also results in the upsizing of the apparatus, it is difficult to apply
these methods to such purposes as home use or PoC (Point of Care). There is another
problem that it is difficult to achieve high accuracy because fine angle adjustment
is required in the order of a millidegree. Besides, since it takes a considerable
time to carry out the angle scan, these methods are difficult to follow a quick reaction
and therefore unsuitable for real-time measurement. Moreover, the measurement range
is also limited because of the limited angle of the light entry, as in the case of
wavelength-variation type.
[0021] Regarding the third and fourth methods, it is unnecessary to carry out the angle
scan of the incident light. However, it is required to point the apex of the wedge
shape of the incident light at the point of measurement so that the light is entered
at the point of measurement on the sensor chip surface from various angles. These
methods are therefore unsuitable for measurement of a large area or multipoint simultaneous
measurement, in which plural points of measurement are measured simultaneously at
a time. Consequently, it is difficult to cope with the increasing packing density
of chips such as a protein chip or a DNA chip and to make full use of the above-described
advantages of the diffraction-grating type sensor chip. Besides, as in the case of
the analysis method in which the measurement is carried out with the constant incident
angle, or also in the cases of the first and second methods, there still remain the
problem that the measurement range is limited and the problem that the optical system
is so complicated the apparatus as such becomes upsized. Moreover, there is another
problem that slight deformation of the reflecting mirror 503 may cause displacements
of the light path and have an impact on accuracy in measurement.
[0022] Regarding the fifth method, as in the cases of the third and fourth methods, analysis
with high accuracy is achieved without the need for carrying out the angle scan of
the incident light and without having to use a driving mechanism. However, the method
has a narrow measurement range (a range within which measurement is allowable) due
to the characteristics of the spectrum shape of the surface plasmon wave, and therefore
is not available for a relatively wide range of concentration. Consequently, there
arises a problem that when the concentration goes outside the measurement range, it
is required to readjust the incident angle and to measure variation in the intensity
of the reflected light again.
[0023] The present invention has been made in view of such problems as mentioned above.
The first object of the invention is to provide a surface plasmon resonance sensor
chip that makes it possible to carry out both real-time measurement and large-area
simultaneous measurement at a time using a optical system having simple constitution,
and to provide a sample analysis method and a sample analysis apparatus using the
same.
[0024] The second object of the invention is to provide a surface plasmon resonance sensor
chip that has a broadened measurement range and makes it possible to handle a sample
having a wide range of concentration when analysis is carried out based on the variation
of the reflected-light intensity, and to provide a sample analysis method and a sample
analysis apparatus using the same.
[0025] The third object of the invention is to provide a surface plasmon resonance sensor
chip that makes it possible to analyze a sample with making full use of a limited
measurement range without the need for readjusting the optical system even when the
dielectric constant or the refractive index has a wide distribution, and to provide
a sample analysis method and a sample analysis apparatus using the same.
[0026] The fourth object of the invention is to provide a surface plasmon resonance sensor
chip that is capable of analyzing a sample using light with a single wavelength under
a simple optical system, and to provide a sample analysis method and a sample analysis
apparatus using the same.
Disclosure of the Invention:
[0027] With the foregoing problems in view, the inventor has the present invention paying
attentions to the following matter. A resonance angle at which surface plasmon resonance
occurs depends on a groove pitch of a diffraction grating viewed from the direction
of incident light. Thus, if the groove pitch, viewed from the direction of incident
light, is distributed (varied) in the diffraction grating, it is possible to accomplish
all the first through fourth objects of the present invention.
[0028] A surface plasmon resonance sensor chip of the present invention comprises: a metal
layer along whose surface a surface plasmon wave can be induced by light irradiation;
and diffraction grating surfaces which are disposed in the vicinity of the metal layer
and on each of which a diffraction grating with a uniform groove orientation and a
uniform groove pitch is formed so as to generate an evanescent wave upon light irradiation.
The groove pitch and the groove orientation of each of the diffraction grating surfaces,
in addition to the angle that each of the diffraction grating surfaces forms with
a predetermined reference plane, are adjusted in such a manner that when the diffraction
grating surfaces are projected onto a predetermined projection plane, the groove orientations
in the projection plane are identical while the groove pitches in the projection plane
are different among the diffraction grating surfaces.
[0029] As a result, the diffraction gratings projected on the projection plane have distributed
groove pitches, and depending on this distribution, the intensity of light reflected
from the surface plasmon resonance sensor chip is also distributed, thus enabling
calculation of resonance angles in real time.
[0030] The followings are two of the preferred embodiments of such a surface plasmon resonance
sensor chip.
[0031] In the first embodiment, a first surface plasmon resonance sensor chip (the first
sensor chip) according to the present invention comprises: a metal layer along whose
surface a surface plasmon wave can be induced by light irradiation; and diffraction
grating surfaces which are disposed in the vicinity of the metal layer and on each
of which a diffraction grating with a uniform groove orientation and a uniform groove
pitch is formed so as to generate an evanescent wave upon light irradiation. Each
of the diffraction grating surfaces is disposed so as to be perpendicular to a specific
plane, which is perpendicular to a predetermined reference plane, and as to form a
predetermined inclination angle with the reference plane, and on each of the diffraction
grating surface, the diffraction grating is formed in such a manner that the groove
orientation is perpendicular to the specific plane.
[0032] With this construction, if a light beam (parallel light) is emitted, from a predetermined
direction, in parallel to the specific plane, incident angles of the irradiated light
with respect to the diffraction grating surfaces are distributed depending on inclination
angles formed by the diffraction gratings with the reference plane. In other words,
when viewed from the incident direction of the incident light, groove pitches of the
diffraction gratings are distributed, and depending on this distribution, the intensity
of light reflected from the surface plasmon resonance sensor chip is also varied.
As a result, it is possible to calculate a resonance angle in real time, based on
the intensity of the light reflected from each diffraction grating surface and a substantial
incident angle of the incident light onto the diffraction grating surface. That is,
using the thus-constructed sensor chip, it is possible to realize effects similar
to those that are achieved when more than one light beam is emitted at different angles
at the same time, with no necessity to perform angle scanning or to eradiate a wedge-formed
light beam.
[0033] As one preferred feature, if two or more diffraction grating surfaces are disposed
along a line parallel to the specific plane, they are arranged in such a manner that
when viewed from a direction parallel to the specific plane, the surfaces are positioned
in decreasing order of the inclination angle that each of the diffraction grating
surfaces forms with the reference plane. As a result, crossing of the light reflected
from the diffraction grating surfaces is prevented, thus facilitating analysis of
the intensity of light reflected from the diffraction grating surfaces.
[0034] As another preferred feature, the diffraction grating surfaces are disposed continuously
so as to form a convex shape whose light-irradiated side bulges out. With this construction,
it is possible to prevent crossing of the light reflected from different positions
on each diffraction grating surface, thus facilitating analysis of the intensity of
light reflected from the diffraction grating surfaces.
[0035] As still another preferred feature, each of the diffraction grating surfaces has
a minimum width, equipped with only one groove alone, and the aggregate of the diffraction
grating surfaces forms a curved surface in an arc shape whose light-irradiated side
bulges out.
[0036] As a further preferred feature, the surface plasmon resonance sensor chip is provided
with more than one diffraction area where diffraction grating surfaces are concentratedly
disposed, and in each of the diffraction areas there are provided diffraction grating
surfaces at different inclination angles. With this preferred feature, it is possible
to detect resonance angles of the separate diffraction grating areas at the same time,
by simply emitting a light beam from a predetermined direction in parallel to the
specific plane. Therefore, with use of binding substances immobilized according to
the diffraction grating areas, simultaneous measurement at multiple positions is easily
available.
[0037] On the above-described first sensor chip, each of the diffraction grating surfaces
is disposed along a sensor surface, which comes in contact with a sample. When the
first sensor chip is used to quantitatively and/or qualitatively analyze the sample,
immobilized binding substances (substances which can capture target species as result
of interaction such as antigen-antibody reaction, complementary DNA bonding, receptor-ligand
interaction, enzyme-substrate interaction) which binds specifically to target species
(chemical species, biochemical species, or biological species, etc.) in the sample
are employed on the sensor surface. In particular, in a case where a sensor chip for
multiple-position simultaneous assessment is used, two or more binding substances
each of which binds to a specific target species in the sample are immobilized in
association with the diffraction areas, thus making it possible to analyze two or
more target species simultaneously.
[0038] It is preferred that two or more binding substances are immobilized for each of the
diffraction grating surfaces.
[0039] It is also preferred that a non-diffraction surface is disposed on the same plane
with the respective one of the diffraction grating surfaces.
[0040] As a preferred feature, the surface plasmon resonance sensor chip has two or more
non-diffraction areas each of which is associated with each of the diffraction area.
Each of the non-diffraction areas has two or more non-diffraction surfaces concentratedly
disposed therein, and none of the non-diffraction surfaces has any diffraction grating.
The inclination angles that the non-diffraction surfaces, included in the non-diffraction
area, form with the reference plane have the same distribution as that of the inclination
angles which the diffraction grating surfaces, included in the associated diffraction
area, form with the reference plane.
[0041] As another preferred feature, each of the diffraction grating surfaces has a reaction
area, within which the binding substance is immobilized, and a non-reaction area,
within which a substance that does not bind to any specific target species in the
sample is immobilized or, alternatively, any substance is not immobilized therein.
[0042] As still another preferred feature, one or more of the diffraction areas each have
a reaction area, in which a binding substance that binds specifically to a target
species in the sample is immobilized. Each of the remaining diffraction areas has
a non-reaction area, in which a' substance that does not bind to any specific target
species in the sample is immobilized or, alternatively, any substance is not immobilized
therein.
[0043] As a further preferred feature, the diffraction grating surfaces are arranged in
a direction perpendicular to the groove orientation, and the sensor chip further includes
a cover for covering the sensor surface and two or more flow channels formed side
by side between the sensor surface and the cover so as to pass along the direction
in which the diffraction grating surfaces are arranged.
[0044] As a still further preferred feature, the sensor chip has a cover for covering the
sensor surface and flow channels disposed side by side between the sensor surface
and the cover, and the diffraction areas are disposed for each of the flow channels.
[0045] The followings are eight of the methods for analyzing a sample using the first sensor
chip. A first analysis method calculates a resonance angle and then analyzes the sample
based on the obtained resonance angle. This method comprises the steps of: making
the sample in contact with the sensor surface while irradiating the sensor surface
with light in parallel to the specific plane at a predetermined incident angle; receiving
light reflected from each of the diffraction grating surfaces and measuring the intensity
of the light reflected by each of the diffraction grating surfaces; calculating a
resonance angle based on both the measured intensity of the reflected light due to
each of the diffraction grating surfaces and the inclination angle that each of the
diffraction grating surfaces forms with the reference plane; and quantitatively and/or
qualitatively analyzing the sample based on the calculated resonance angle.
[0046] With this method, since the resonance angle is calculated instantaneously without
necessity to perform angle scanning, thus enabling real-time measurement, it is possible
to calculate the resonance angle without necessity to emit a wedge-shaped light beam,
thereby making it possible to perform measurement in a large area all at once. Accordingly,
using a sensor chip for multiple-position simultaneous assessment, on which chip there
are provided diffraction grating areas where diffraction grating surfaces are arranged
in a concentrated manner, it is also possible to perform such multiple-position simultaneous
assessment in real time. In this case, partly since the optical axis of a light source
is not required to change, and partly since parallel light can be used, a simple optical
system can be employed. It is to be noted that the above steps can be performed in
the order of the foregoing description, or alternatively, they can be performed simultaneously.
In the latter case, particularly, it is possible to monitor in real time how target
species in the sample bind to binding substances.
[0047] The above-described analysis method is realized by an analysis apparatus constructed
as follows. The apparatus comprises: a holding means for holding such a first surface
plasmon resonance sensor chip with the sensor surface being in contact with the sample;
a light irradiating means for irradiating the sensor surface with light in parallel
to the specific plane at a predetermined incident angle in a state where the surface
plasmon resonance sensor chip is held by the holding means; a light receiving means
for receiving the light reflected from each of the diffraction grating surfaces ;
a measuring means for measuring the intensity of the light reflected by each of the
diffraction grating surfaces and received by the light receiving means. The apparatus
further comprises a calculating means and an analyzing means, for analyzing the sample
based on the reflected light received by the light receiving means. The calculating
means calculates a resonance angle based on both the intensity, measured by the measuring
means, of the reflected light due to each of the diffraction grating surfaces and
the inclination angle that each of the diffraction grating surfaces forms with the
reference plane; the analyzing means quantitatively and/or qualitatively analyzes
the sample based on the resonance angle calculated by the calculating means.
[0048] A second analysis method calculates a resonance angle and then analyzes the sample
based on the obtained resonance angle. The method employs a surface plasmon resonance
sensor chip with non-diffraction surfaces formed thereon. Each of the non-diffraction
surfaces does not have any diffraction grating and is disposed along the sensor surface
on the same plane with the respective one of the diffraction grating surfaces. This
method comprises the steps of: making a sample in contact with the sensor surface
while irradiating the sensor surface with light in parallel to the specific plane
at a predetermined incident angle; receiving light reflected from the sensor surface
and measuring the intensity of the light reflected by each of the diffraction grating
surfaces; correcting the measured intensity of the reflected light due to each of
the diffraction grating surfaces with consideration given to the intensity of the
light reflected by each of the non-diffraction surfaces; calculating a resonance angle
at which a resonance phenomenon of the evanescent wave and the surface plasmon wave
occurs, based on both the measured intensity of reflected light due to each of the
diffraction grating surfaces and the inclination angle that each of the diffraction
grating surfaces forms with the reference plane; and quantitatively and/or qualitatively
analyzing the sample based on the calculated resonance angle.
[0049] Here, a non-diffraction surface reflecting the light that is to be considered for
correction must be inclined with respect to the reference plane at the same angle
as that at which an object diffraction grating surface reflecting the light that is
to be corrected is inclined with respect to the reference plane. Therefore, if such
a non-diffraction grating surface and the object diffraction grating surface are not
disposed on the same plane, it is required to specify a non-diffraction surface that
corresponds to the object diffraction grating surface, so that correction can be performed
in consideration of the reflected light coming from the specified non-diffraction
surface.
[0050] With this method, not only similar effects to those of the first analysis method
but also the following advantages can be attained. Since the intensity of light reflected
from each diffraction grating surface is corrected in consideration of the intensity
of light reflected from a non-diffraction surface, it is possible to correct errors
in the intensity of reflected light caused by differences in surface properties among
the diffraction grating surfaces.
[0051] In this instance, the above-mentioned surface properties are defined as factors,
out of those relating to the surface of the sensor chip except a resonance phenomenon
of the evanescent wave and the surface plasmon wave, that weaken the intensity of
reflected light from a sensor chip. Such example factors are, for instance, cloudy
sample solutions causing a scattering of light; substances in sample solutions that
absorb incident light; and slight displacement, such as distortion, deflection, swelling,
and contraction, from an ideal surface. In addition, if a constituent of the sample
is nonspecifically adsorbent to the surface of the sensor chip, incident light can
be scattered or absorbed by this substance, the intensity of the reflected light from
the sensor chip being thereby decreased.
[0052] This analysis method is realized by an analysis apparatus constructed as follows.
The apparatus comprises: a holding means for holding such the above mentioned first
surface plasmon resonance sensor chip with the sensor surface being in contact with
a sample; a light irradiating means for irradiating the sensor surface with light
in parallel to the specific plane at a predetermined incident angle in a state where
the surface plasmon resonance sensor chip is held by the holding means; a light receiving
means for receiving the light reflected from the diffraction grating surfaces; a measuring
means for measuring the intensity of the light received by the light receiving means.
The apparatus further comprises a correcting means, a calculating means, and an analyzing
means, for analyzing the sample based on the reflected light received by the light
receiving means. The correcting means corrects the intensity of reflected light due
to each of the diffraction grating surfaces with consideration given to the intensity
of the reflected light due to the non-diffraction surface; a calculating means calculates
a resonance angle based on both the intensity, corrected by the correcting means,
of the reflected light due to each of the diffraction grating surfaces and the inclination
angle that each of the diffraction grating surfaces forms with the reference plane;
and the analyzing means quantitatively and/or qualitatively analyzes the sample based
on the resonance angle calculated by the calculating means.
[0053] A third analysis method calculates a resonance angle and then analyzes the sample
based on the obtained resonance angle. The method employs a surface plasmon resonance
sensor chip that has a reaction area, within which the binding substance is immobilized,
and a non-reaction area, within which a substance that does not bind specifically
to any target species in the sample is immobilized or, alternatively, any substance
is not immobilized. The analysis method comprises the steps of: making a sample in
contact with the sensor surface while irradiating the sensor surface with light in
parallel to the specific plane at a predetermined incident angle; receiving light
reflected from the sensor surface and measuring the intensity of the light reflected
by each of the diffraction grating surfaces; calculating, for each of the reaction
area and the non-reaction area, a resonance angle at which a resonance phenomenon
of the evanescent wave and the surface plasmon wave occurs, based on both the measured
intensity of the reflected light due to each of the diffraction grating surface and
the inclination angle that each of the diffraction grating surfaces forms with the
reference plane; and after correcting the resonance angle of the reaction area with
consideration given to the resonance angle of the non-reaction area, quantitatively
and/or qualitatively analyzing the sample based on the corrected resonance angle of
the reaction area.
[0054] With this method, not only similar effects to those of the first analysis method
but also the following advantages can be attained. Since the resonance angle in a
reaction area is corrected based on the resonance angle in a non-reaction area, it
is possible to accurately analyze changes in target species caused by reactions.
[0055] As for the correction performed according to this method, if a resonance angle can
be obtained, it is not always necessary that the reaction area be inclined with respect
to the reference plane at the same angle as that at which a non-reaction area is inclined
with respect to the reference plane. This is because it is an amount of reaction,
not the intensity of reflected light, which is to be corrected here based on a shift
in the resonance angle due to other factors than specific target reactions to be detected.
[0056] The above-described analysis method is realized by an analysis apparatus constructed
as follows. The apparatus comprises: a holding means for holding such a first plasmon
resonance sensor chip with the sensor surface being in contact with the sample; a
light irradiating means for irradiating the sensor surface with light in parallel
to the specific plane at a predetermined incident angle in a state where the surface
plasmon resonance sensor chip is held by the holding means; a light receiving means
for receiving the light reflected from the sensor surface; and a measuring means for
measuring the intensity of the light reflected by each of the diffraction grating
surfaces and received by the light receiving means. The apparatus further comprises
a calculating means and an analyzing means, for analyzing the sample based on the
reflected light received by the light receiving means. The calculating means calculates,
for each of the reaction area and the non-reaction area, a resonance angle at which
a resonance phenomenon of the evanescent wave and the surface plasmon wave occurs,
based on the intensity, measured by the measuring means, of the reflected light due
to each of the diffraction grating surfaces and the inclination angle that each of
the diffraction grating surfaces forms with the reference plane; and the analyzing
means quantitatively and/or qualitatively analyzes the sample based on a corrected
resonance angle of the reaction area, which is corrected with consideration given
to the resonance angle of the non-reaction area.
[0057] A fourth analysis method calculates a resonance angle and then analyzes the sample
based on the obtained resonance angle. The method employs a surface plasmon resonance
sensor chip on which the diffraction grating surfaces are arranged in a direction
perpendicular to the groove orientation. The sensor chip has one of the following
two types of constructions: (1) the sensor chip has a cover for covering the sensor
surface, and channels formed side by side between the sensor surface and the cover
so as to pass along the direction in which the diffraction grating surfaces are arranged;
(2) the sensor chip has a cover for covering the sensor surface, and flow channels
disposed side by side between the sensor surface and the cover, and the diffraction
areas are disposed for each of the flow channels. The analysis method comprises the
steps of: assigning different samples one to each of the flow channels, and letting
each of the samples flow through the respective flow channel while irradiating the
sensor surface with light in parallel to the specific plane at a predetermined incident
angle; receiving the light reflected from the sensor surface and measuring the intensity
of the light reflected by each of the diffraction grating surfaces; calculating, for
each sample flowing through the respective flow channel, a resonance angle at which
a resonance phenomenon of the evanescent wave and the surface plasmon wave occurs,
based on both the measured intensity of the reflected light due to each of the diffraction
grating surfaces and the inclination angle that each of the diffraction grating surfaces
forms with the reference plane; and quantitatively and/or qualitatively analyzing
each sample flowing through the respective flow channel, based on the calculated resonance
angle for each of the flow channels.
[0058] With this method, not only similar effects to those of the first analysis method
but also the following advantages can be attained. Different kinds of samples can
be analyzed simultaneously, thus realizing effective analysis. In addition, since
the different samples are analyzed under an identical condition, it is possible to
make the difference among the samples clear.
[0059] This analysis method is realized by an analysis apparatus constructed as follows.
The apparatus comprises: a holding means for holding such a first surface plasmon
resonance sensor chip; a sample introducing means for assigning different samples
one to each of the flow channels, and for introducing each of the samples into the
respective flow channel in a state where the surface plasmon resonance sensor chip
is held by the holding means; a light irradiating means for irradiating the sensor
surface with light in parallel to the specific plane at a predetermined incident angle
in a state where each sample is introduced into the respective flow channel by the
sample introducing means; a light receiving means for receiving the light reflected
from the sensor surface; a measuring means for measuring the intensity of the light
reflected by each of the diffraction grating surface and received by the light receiving
means. The apparatus further includes a calculating means and an analyzing means,
for analyzing the sample based on the reflected light received by the light receiving
means. The calculating means calculates a resonance angle at which a resonance phenomenon
of the evanescent wave and the surface plasmon wave occurs for each of the flow channels,
based on the intensity, measured by the measuring means, of the reflected light due
to each of the diffraction grating surfaces and the inclination angle that each of
the diffraction grating surfaces forms with the reference plane; and the analyzing
means quantitatively and/or qualitatively analyzes each sample flowing through the
respective flow channel, based on the resonance angle calculated by the calculating
means.
[0060] A fifth analysis method measures the variation in the intensity of reflected light
and then analyzes a sample based on the variation amount. The method comprises the
steps of: making the sample in contact with the sensor surface while irradiating the
sensor surface with light in parallel to the specific plane at a predetermined incident
angle; receiving light reflected from each of the diffraction grating surfaces and
measuring the intensity of the light reflected by each of the diffraction grating
surfaces; determining the variation between the measured intensity of the reflected
light due to each of the diffraction grating surfaces and the intensity of the light
reflected when any sample is not in contact with the sensor surface; and selecting
a diffraction grating surface whose determined variation of the reflected light intensity
is within a predetermined allowable range for determination, and quantitatively and/or
qualitatively analyzing the sample based on the variation of the reflected light intensity
of the selected diffraction grating surface.
[0061] With this method, even when a wide range of sample concentrations are used, it is
not required to readjust the optical system in such a manner that measurement results
fall within a measurement range, the measurement range thereby being virtually enlarged.
In this case, also, the above steps can be performed in the order of the foregoing
description, or alternatively, they can be performed simultaneously.
[0062] The above-described analysis method is realized by an analysis apparatus constructed
as follows. The apparatus comprises: a holding means for holding such a first surface
plasmon resonance sensor chip with the sensor surface being in contact with the sample;
a light irradiating means for irradiating the sensor surface with light in parallel
to the specific plane at a predetermined incident angle in a state where the surface
plasmon resonance sensor chip is held by the holding means; a light receiving means
for receiving the light reflected from each of the diffraction grating surfaces; a
measuring means for measuring the intensity of the light reflected by each of the
diffraction grating surfaces and received by the light receiving means. The apparatus
further comprises a determining means and an analyzing means, for analyzing the sample
based on the reflected light received by the light receiving means. The determining
means determines the variation between the intensity, measured by the measuring means,
of the reflected light due to each of the diffraction grating surfaces and the intensity
of the light reflected when any sample is not in contact with the sensor surface;
and the analyzing means selects a diffraction grating surface whose variation, determined
by the determining means, of the reflected light intensity is within a predetermined
allowable range for determination, and quantitatively and/or qualitatively analyzes
the sample based on the variation of the reflected light intensity of the selected
diffraction grating surface.
[0063] A sixth analysis method measures the variation in the intensity of reflected light
and then analyzes a sample based on the variation amount. The method employs a surface
plasmon resonance sensor chip comprising non-diffraction surfaces with no diffraction
grating formed thereon. Each of the non-diffraction surfaces is disposed on the same
plane along the sensor surface on the same plane with the respective one of the diffraction
grating surfaces. This method comprises the steps of: making the sample in contact
with the sensor surface while irradiating the sensor surface with light in parallel
to the specific plane at a predetermined incident angle; receiving light reflected
from the sensor surface and measuring the intensity of the light reflected by each
of the diffraction grating surfaces; correcting the intensity of the reflected light
due to each of the diffraction grating surfaces with consideration given to the intensity
of the reflected light due to each of the non-diffraction surface; determining the
variation between the corrected intensity of the reflected light due to each of the
diffraction grating surface and the intensity of the light reflected when any sample
is not in contact with the sensor surface; and selecting a diffraction grating surface
whose determined variation of the reflected light intensity is within a predetermined
allowable range (determination range) for determination, and quantitatively and/or
qualitatively analyzing the sample based on the variation of the reflected light intensity
of the selected diffraction grating surface.
[0064] Here, as in the case of the second analysis method, a non-diffraction surface reflecting
the light that is to be considered on correction must be inclined with respect to
the reference plane at the same angle as that at which an object diffraction grating
surface reflecting the light to be corrected is inclined with respect to the reference
plane. Therefore, if such a non-diffraction grating surface and the object diffraction
grating surface are not disposed on the same plane, it is required to specify a non-diffraction
surface that corresponds to the object diffraction grating surface, so that correction
can be performed in consideration of the reflected light coming from the specified
non-diffraction surface.
[0065] With this method, not only similar effects to those of the fifth analysis method
but also the following advantages can be attained. Since the intensity of light reflected
from the diffraction grating surfaces is corrected in consideration of the intensity
of light reflected from the non-diffraction surfaces, it is possible to correct errors
in the intensity of reflected light caused by differences in surface properties among
the diffraction grating surfaces.
[0066] This analysis method is realized by an analysis apparatus constructed as follows.
The apparatus comprises: a holding means for holding such a first surface plasmon
resonance sensor chip with the sensor surface being in contact with a sample; a light
irradiating means for irradiating the sensor surface with light in parallel to the
specific plane at a predetermined incident angle in a state where the surface plasmon
resonance sensor chip is held by the holding means; a light receiving means for receiving
the light reflected from each of the diffraction grating surfaces; and a measuring
means for measuring the intensity of the light reflected by each of the diffraction
grating surfaces and received by the light receiving means. The apparatus further
comprises a correcting means, a determining means, and an analyzing means, for analyzing
the sample based on the reflected light received by the light receiving means. The
correcting means corrects the intensity of reflected light due to each of the diffraction
grating surface with consideration given to the intensity of the reflected light due
to the non-diffraction surface; the determining means for determining the variation
between the intensity, corrected by the correcting means, of the reflected light due
to each of the diffraction grating surfaces and the intensity of the light reflected
when any sample is not in contact with the sensor surface; and an analyzing means
for selecting a diffraction grating surface whose variation, determined by the determining
means, of the reflected light intensity is within a predetermined allowable range
for determination, and for quantitatively and/or qualitatively analyzing the sample
based on the variation of the reflected light intensity of the selected diffraction
grating surface.
[0067] A seventh analysis method measures variation in the intensity of reflected light
and then analyzes a sample based on the variation amount. The method employs a surface
plasmon resonance sensor chip that has a reaction area, within which a binding substance
is immobilized, and a non-reaction area, within which a substance that does not bind
specifically to any target species in the sample is immobilized or, alternatively,
any substance is not immobilized. The method comprises the steps of: making a sample
in contact with the sensor surface while irradiating the sensor surface with light
in parallel to the specific plane at a predetermined incident angle; receiving the
light reflected from each of the diffraction grating surfaces and measuring the intensity
of the light reflected by each of the diffraction grating surfaces; determining, for
each of the reaction areas and the non-reaction areas, the variation between the measured
intensity of the reflected light due to each of the diffraction grating surfaces and
the intensity of the light reflected when any sample is not in contact with the sensor
surface; and selecting, for each of the reaction areas and the non-reaction areas,
a diffraction grating surface whose determined variation of the reflected-light intensity
is within a predetermined allowable range for determination, and quantitatively and/or
qualitatively analyzing the sample based on both the selected variation of the reflected
light intensity of the reaction area and the selected variation of the reflected light
intensity of the non-reaction area.
[0068] As in the case of the third analysis method, if a resonance angle can be obtained,
it is not always necessary that the reaction area be inclined with respect to the
reference plane at the same angle as that at which a non-reaction area is inclined
with respect to the reference plane. This is because it is an amount of reaction,
not the intensity of reflected light, which is to be corrected here based on a shift
in the resonance angle due to other factors than specific target reactions that are
to be detected.
[0069] With this method, not only similar effects to those of the fifth analysis method
but also the following advantages can be attained. Since the resonance angle in a
reaction area is corrected based on the resonance angle in a non-reaction area, it
is possible to accurately analyze change in target species caused by reactions.
[0070] The above-described analysis method is realized by an analysis apparatus constructed
as follows. The apparatus comprises: a holding means for holding such a first surface
plasmon resonance sensor chip with the sensor surface being in contact with a sample;
a light irradiating means for irradiating the sensor surface with light in parallel
to the specific plane at a predetermined incident angle in a state where the surface
plasmon resonance sensor chip is held by the holding means; a light receiving means
for receiving the light reflected from each of the diffraction grating surfaces; and
a measuring means for measuring the intensity of the light reflected by each of the
diffraction grating surfaces and received by the light receiving means. The apparatus
further comprises a determining means and an analyzing means, for analyzing the sample
based on the reflected light received by the light receiving means. The determining
means determines, for each of the reaction areas and the non-reaction areas, the variation
between the intensity, measured by the measuring means, of the reflected light due
to each of the diffraction grating surfaces and the intensity of the light reflected
when any sample is not in contact with the sensor surface; and the analyzing means
selects a diffraction grating surface whose variation, determined by the determining
means, of the reflected light intensity is within a predetermined allowable range
for determination, and quantitatively and/or qualitatively analyzes the sample based
on both the variation of the reflected light intensity of the selected reaction area
and the variation of the reflected light intensity of the selected non-reaction area.
[0071] An eighth analysis method measures the variation in the intensity of reflected light
and then analyzes a sample based on the variation amount. The method employs a surface
plasmon resonance sensor chip on which the diffraction grating surfaces are arranged
in a direction perpendicular to a groove orientation. The chip has one of the following
two types of constrictions: (1) the chip further includes a cover for covering the
sensor surface, and flow channels formed side by side between the sensor surface and
the cover so as to pass along the direction in which the diffraction grating surfaces
are arranged; (2) the chip has a cover for covering the sensor surface, and flow channels
disposed side by side between the sensor surface and the cover, and the diffraction
areas are disposed for each of the flow channels. This analysis method comprises the
steps of: assigning different samples one to each of the plural flow channels, and
letting each of the samples flow through the respective flow channel while irradiating
the sensor surface with light in parallel to the specific plane at a predetermined
incident angle; receiving light reflected from the sensor surface and measuring the
intensity of the light reflected by each of the diffraction grating surfaces; determining
the variation between the measured intensity of the reflected light due to each of
the diffraction grating surfaces and the intensity of the light reflected when any
sample does not flow through the flow channels; and selecting, for each of the flow
channels, a diffraction grating surface whose determined variation of the reflected
light intensity is within a predetermined allowable range for determination, and quantitatively
and/or qualitatively analyzing each sample flowing through the respective flow channel,
based on the variation of the reflected light intensity of the diffraction grating
surface selected for each of the flow channels.
[0072] With this method, not only similar effects to those of the fifth analysis method
but also the following advantages can be attained. It is possible to analyze two or
more samples simultaneously, thus realizing effective analysis. Additionally, since
the different samples are analyzed under an identical condition, it is possible to
make the difference among the samples clear.
[0073] This analysis method is realized by an analysis apparatus constructed as follows.
The apparatus comprises: a holding means for holding such a first surface plasmon
resonance sensor chip; a sample introducing means for assigning different samples
one to each of the flow channels, and for introducing each of the samples into the
respective flow channel in a state where the surface plasmon resonance sensor chip
is held by the holding means; a light irradiating means for irradiating the sensor
surface with light in a predetermined direction in a state where each sample is introduced
into the respective flow channel by the sample introducing means; a light receiving
means for receiving light reflected from each of the diffraction grating surfaces;
and a measuring means for measuring the intensity of the light reflected by each of
the diffraction grating surface and received by the light receiving means. The apparatus
further comprises a determining means and an analyzing means, for analyzing the sample
based on the light reflected received by the light receiving means. The determining
means determines the variation between the intensity of the light reflected by each
of the diffraction grating surfaces and received by the light receiving means and
the intensity of the light reflected when any sample is not flowing through the flow
channels; and the analyzing means selects, for each of the flow channels, a diffraction
grating surface whose variation, determined by the determining means, of the reflected
light intensity is within a predetermined allowable range for determination, and quantitatively
and/or qualitatively analyzes each sample flowing through the respective flow channel,
based on the variation of the reflected light intensity of the diffraction grating
surface selected for each of the flow channel.
[0074] As a preferred feature, the above method further comprises the step of separating
the sample by physical and/or chemical action prior to introducing the sample to the
surface plasmon resonance sensor chip.
[0075] This makes it possible to appropriately remove impurities, if any, other than target
species contained in the sample before analysis, so that only pure target species
can be subject to the analysis. As a consequence, the accuracy of the analysis is
improved.
[0076] At that time, if combined with detecting techniques (absorbance detection, fluorescence
detection, chemiluminescence detection, differential refract meter detection, electrochemical
detection, etc.) that are commonly used in these analyzing means, it is possible to
perform the following determination at the same time: determination of the amount
of various substances being present; and determination of target species by measuring
reactions unique to the target species.
[0077] This analysis method is implemented by an analysis apparatus which has, in addition
to the construction as described above, a sample separating means for separating the
sample by physical and/or chemical action prior to introducing the sample to the surface
plasmon resonance sensor chip.
[0078] The following separation techniques preferably serve as such sample separating means:
liquid chromatography; HPLC (High Performance Liquid Chromatography); capillary electrophoresis;
microchip electrophoresis; and methods using flow injection or microchannels.
[0079] As a preferred feature, if the target species is a light-emitting substance, the
method further comprises the step of detecting light emitted from the light-emitting
substance that is bound to the binding substance prior to light-irradiating the sensor
surface or, alternatively, after light-irradiating the sensor surface and receiving
the reflected light. In the step of quantitatively and/or qualitatively analyzing,
the sample is analyzed with consideration given to the detection result of the emitted
light.
[0080] With this feature, light-emitting phenomena as well as surface plasmon resonance
can be utilized in the analysis. The extremely high sensitivity of light-emitting
phenomena, such as fluorescence and phosphor, enables detection of minute reactions.
[0081] This analyzing means can be implemented by an analysis apparatus that has, in addition
to the construction as described above, the following construction: when the target
species is a light-emitting substance, the light receiving means is operable to detect
light emitted from the light-emitting substance that is bound to the binding substance,
and the analyzing means is operable to quantitatively and/or qualitatively analyze
the sample with consideration given to the detection result of the light emission
by the light receiving means.
[0082] Next, a second surface plasmon resonance chip (second sensor chip) according to the
present invention comprises: a metal layer along whose surface a surface plasmon wave
can be induced by light irradiation; and diffraction grating surfaces which are disposed
in the vicinity of the metal layer and on each of which a diffraction grating with
a uniform groove orientation and a uniform groove pitch is formed so as to generate
an evanescent wave upon light irradiation. Each of the diffraction grating surfaces
is disposed so as to be parallel to a predetermined reference plane, and on each of
the diffraction grating surfaces, the diffraction grating is formed in such a manner
that the groove orientations are identical while the groove pitches are different
among the diffraction grating surfaces.
[0083] With this construction, when light (parallel light) is emitted from a predetermined
direction, an evanescent wave occurs which has the number of waves and an angular
frequency corresponding to a groove pitch of each diffraction grating surface. As
a result, resonance phenomena occur at two or more resonance points with different
angular frequencies for one single surface plasmon wave. It is thus possible to calculate
more than one resonance points in real time, based on the intensity of the light reflected
from the diffraction grating surfaces and groove pitches on the diffraction grating
surfaces. In other words, such a sensor chip makes it possible to detect more than
one resonance point even using a simple optical system.
[0084] Such a second sensor chip has diffraction grating surfaces formed along a sensor
surface, which comes in contact with a sample. If such a second sensor chip is used
in quantitative and/or qualitative analysis of a sample, a binding substance (a substance
which captures a target species by antigen-antibody reaction, complementary DNA bonding,
receptor-ligand interaction, enzyme-substrate interaction) that binds specifically
to a target species (chemical, biochemical, or biological species) in the sample is
immobilized on the sensor surface, for each of the diffraction grating surfaces. In
particular, in case of a sensor chip for a multiple-point simultaneous measurement,
two or more kinds of binding substances are immobilized for each of the diffraction
grating surfaces.
[0085] As a preferred feature, on the sensor surface there are provided non-diffraction
surfaces with no diffraction grating formed thereon. Each of the non-diffraction surfaces
is disposed along the sensor surface on the same plane with the respective one of
the diffraction grating surfaces.
[0086] As another preferred feature, each of the diffraction grating surfaces has a reaction
area, within which the binding substance is immobilized, and a non-reaction area,
within which a substance that does not bind specifically to any target species in
the sample is immobilized or, alternatively, any substance is not immobilized.
[0087] As still another preferred feature, the diffraction grating surfaces are arranged
in a direction perpendicular to the groove orientation, and the sensor chip further
comprises a cover for covering the sensor surface, and flow channels formed side by
side between the sensor surface and the cover so as to pass along the direction in
which the diffraction grating surfaces are arranged.
[0088] As a further preferred feature, the diffraction grating surfaces are arranged in
a direction perpendicular to the groove orientation. The sensor chip further comprises:
a cover for covering the sensor surface; and flow channels formed side by side between
the sensor surface and the cover so as to pass along the direction in which the diffraction
grating surfaces are arranged. Along each of the flow channels, each of the diffraction
grating surfaces has a reaction area, within which the binding substance is immobilized,
and a non-reaction area, within which a substance that does not bind specifically
to any target species in the sample is immobilized or, alternatively, any substance
is not immobilized.
[0089] The followings are ten of the methods for analyzing a sample using such a second
sensor chip.
[0090] A first analysis method calculates a groove pitch at which resonance occurs and then
quantitatively and/or qualitatively analyzes a sample based on the obtained groove
pitch. This method comprises the steps of: making a sample in contact with the sensor
surface while irradiating the sensor surface with light at a predetermined incident
angle; receiving light reflected from each of the diffraction grating surfaces and
measuring the intensity of the light reflected by each of the diffraction grating
surfaces; correcting the intensity of the reflected light due to each of the diffraction
grating surfaces with consideration given to the intensity of the light reflected
by the respective non-diffraction surface; identifying a groove pitch at which a resonance
phenomenon of the evanescent wave and the surface plasmon wave occurs, based on the
corrected intensity of the reflected light due to each of the diffraction grating
surfaces; and quantitatively and/or qualitatively analyzing the sample based on the
identified groove pitch.
[0091] With this analysis method, it is possible to calculate more than one resonance point
instantaneously, thus enabling real-time measurement. In addition, even if wide ranges
of dielectric constant distribution and refractive index distribution are found, it
is still possible to analyze a sample without readjusting an optical system. Accordingly,
using a sensor chip for multiple-position simultaneous analysis, on which chip there
are provided diffraction grating areas where diffraction grating surfaces are arranged
in a concentrated manner, it is possible to perform such multiple-position simultaneous
assessment in real time. In addition, partly since the optical axis of a light source
is not required to change, and partly since parallel light can be used, it is possible
to employ a simple optical system. It is to be noted that the above steps can be performed
in the order of the foregoing description, or alternatively, they can be performed
simultaneously. In the latter case, particularly, it is possible to monitor in real
time how target species bind to binding substances.
[0092] The above-described analysis method is realized by an analysis apparatus constructed
as follows. The apparatus comprises: a holding means for holding such a second surface
plasmon resonance sensor chip with the sensor surface being in contact with a sample;
a light irradiating means for irradiating the sensor surface with light from a predetermined
direction in a state where the surface plasmon resonance sensor chip is held by the
holding means; a light receiving means for receiving the light reflected from the
sensor surface; a measuring means for measuring the intensity of the light reflected
by each of the diffraction grating surfaces and received by light receiving means.
The apparatus further comprises an analyzing means for analyzing the sample based
on the reflected light received by the light receiving means. The analyzing means
identifies a groove pitch at which a resonance phenomenon of the evanescent wave and
the surface plasmon wave occurs, based on the intensity, measured by the measuring
means, of the reflected light due to each of the diffraction grating surfaces, and
quantitatively and/or qualitatively analyzes the sample based on the identified groove
pitch.
[0093] A second analysis method calculates a groove pitch at which resonance occurs and
then quantitatively and/or qualitatively analyzes a sample based on the obtained groove
pitch. The method employs a surface plasmon resonance sensor chip comprising non-diffraction
surfaces with no diffraction grating formed thereon, each of which non-diffraction
surfaces is disposed on the same plane along the sensor surface on the same plane
with the respective one of the diffraction grating surfaces. This method comprises
the steps of: making a sample in contact with the sensor surface while irradiating
the sensor surface with light at a predetermined incident angle; receiving light reflected
from the sensor surface and measuring the intensity of the light reflected by each
of the diffraction grating surfaces; correcting the intensity of the reflected light
due to each of the diffraction grating surfaces with consideration given to the intensity
of the light reflected by the respective non-diffraction surface; identifying a groove
pitch at which a resonance phenomenon of the evanescent wave and the surface plasmon
wave occurs, based on the corrected intensity of the reflected light due to each of
the diffraction grating surfaces; and quantitatively and/or qualitatively analyzing
the sample based on the identified groove pitch.
[0094] With this method, not only similar effects to those of the first analysis method
but also the following advantages are attained. Since the intensity of light reflected
from the diffraction grating surfaces is corrected in consideration of the intensity
of light reflected from the non-diffraction surfaces, it is possible to correct errors
in the intensity of reflected light caused by differences in surface properties among
the diffraction grating surfaces.
[0095] In this instance, the above-mentioned surface properties are defined as factors,
out of those relating to the surface of the sensor chip, that weaken the intensity
of light reflected from a sensor chip. Such example factors are, for instance, cloudy
sample solutions causing a scattering of light, substances in sample solutions that
absorb incident light, and displacement, such as distortion, deflection, swelling,
and contraction, from an ideal surface. In addition, if a constituent of the sample
is nonspecifically adsorbent to the surface of the sensor chip, incident light can
be scattered or absorbed by this substance, the intensity of the light reflected from
the sensor chip being thereby decreased.
[0096] This analysis method is realized by an analysis apparatus constructed as follows.
The apparatus comprises: a holding means for holding such a second surface plasmon
resonance sensor chip with the sensor surface being in contact with a sample; a light
irradiating means for irradiating the sensor surface with light from a predetermined
direction in a state where the surface plasmon resonance sensor chip is held by the
holding means; a light receiving means for receiving light reflected from each of
the diffraction grating surfaces; a measuring means for measuring the intensity of
the light reflected by each of the diffraction grating surface and received by the
light receiving means. The apparatus further comprises a correcting means and an analyzing
means, for analyzing the sample based on the reflected light received by the light
receiving means. The correcting means corrects the intensity of the reflected light
due to each of the diffraction grating surfaces with consideration given to the intensity
of the reflected light due to the respective non-diffraction surface; and the analyzing
means identifies a groove pitch at which a resonance phenomenon of the evanescent
wave and the surface plasmon wave occurs, based on the intensity, corrected by the
correcting means, of the reflected light due to each of the diffraction grating surfaces,
and quantitatively and/or qualitatively analyzes the sample based on the identified
groove pitch.
[0097] A third analysis method calculates a groove pitch at which resonance occurs and then
quantitatively and/or qualitatively analyzes a sample based on the obtained groove
pitch. The method employs a surface plasmon resonance sensor chip formed as follows:
each of the diffraction grating surfaces has a reaction area, within which the binding
substance is immobilized, and a non-reaction area, within which a substance that does
not bind specifically to any target species in the sample is immobilized or, alternatively,
any substance is not immobilized. This method comprises the steps of: making a sample
in contact with the sensor surface while irradiating the sensor surface with light
at a predetermined incident angle; receiving the light reflected from the sensor surface
and measuring the intensity of the light reflected by each of the diffraction grating
surfaces; identifying, for each of the reaction areas and the non-reaction areas,
a groove pitch at which a resonance phenomenon of the evanescent wave and the surface
plasmon wave occurs, based on the measured intensity of the reflected light due to
each of the diffraction grating surfaces; and quantitatively and/or qualitatively
analyzing the sample based on the groove pitch identified for each of the reaction
areas and the non-reaction areas.
[0098] With this analysis method, not only similar effects to those of the first analysis
method but also the following advantages can be attained. Since the analysis is performed
based on groove pitches of the reaction areas and those of the non-reaction areas,
it is possible to accurately analyze changes in target species caused by reactions.
[0099] This analysis method is realized by an analysis apparatus constructed as follows.
The apparatus comprises: a holding means for holding such a second surface plasmon
resonance sensor chip with the sensor surface being in contact with a sample; a light
irradiating means for irradiating the sensor surface with light at a predetermined
incident angle in a state where the surface plasmon resonance sensor chip is held
by the holding means; a light receiving means for receiving light reflected from each
diffraction grating surface; and a measuring means for measuring the intensity of
the light received by the light receiving means. The apparatus further comprises an
analyzing means for analyzing the sample based on the light received by the light
receiving means. The analyzing means identifies, for each of the reaction area and
the non-reaction area, a groove pitch at which a resonance phenomenon of the evanescent
wave and the surface plasmon wave occurs, based on the intensity, measured by the
measuring means, of the reflected light due to each of the diffraction grating surface,
and quantitatively and/or qualitatively analyzes the sample based on the groove pitch
identified for each of the reaction areas and the non-reaction areas.
[0100] A fourth analysis method calculates a groove pitch at which resonance occurs and
then quantitatively and/or qualitatively analyzes a sample based on the obtained groove
pitch. The method employs a surface plasmon resonance sensor chip formed as follows.
On the sensor ship, the diffraction grating surfaces are arranged in a direction perpendicular
to the groove orientation, and the sensor chip further comprises a cover for covering
the sensor surface and flow channels formed side by side between the sensor surface
and the cover so as to pass along the direction in which the diffraction grating surfaces
are arranged. This method comprises the steps of: assigning different samples one
to each of the flow channels, and letting each of the samples flow through the respective
flow channel while irradiating the sensor surface with light at a predetermined incident
angle; receiving light reflected from the sensor surface and measuring the intensity
of the light reflected by each of the diffraction grating surfaces; identifying, for
each of the flow channels, a groove pitch at which the resonance phenomenon of the
evanescent wave and the surface plasmon wave occurs, based on the measured intensity
of the reflected light due to each of the diffraction grating surfaces; and quantitatively
and/or qualitatively analyzing each sample flowing through the respective flow channel,
based on the groove pitch identified for each of the flow channels.
[0101] With this analysis method, not only similar effects to those of the first analysis
method but also the following advantages can be attained. More than one target species
is simultaneously analyzed, thus realizing effective analysis. In addition, since
more than one type of target species can be analyzed under an identical condition,
it is possible to make the difference among the target species clear.
[0102] This analysis method is realized by an analysis apparatus constructed as follows.
The apparatus comprises: a holding means for holding such a second surface plasmon
resonance sensor chip; a sample introducing means for assigning different samples
one to each of the plural flow channels, and for introducing each of the samples into
the respective flow channel in a state where the surface plasmon resonance sensor
chip is held by the holding means; a light irradiating means for irradiating the sensor
surface with light from a predetermined direction in a state where each sample is
introduced into the respective flow channel by the sample introducing means; a light
receiving means for receiving light reflected from each of the diffraction grating
surfaces; and a measuring means for measuring the intensity of the light reflected
by each of the diffraction grating surface and received by the light receiving means.
The apparatus further comprises an analyzing means for analyzing the sample based
on the light received by the light receiving means. The analyzing means identifies,
for each of the flow channels, a groove pitch at which a resonance phenomenon of the
evanescent wave and the surface plasmon wave occurs, based on the intensity, measured
by the measuring means, of the reflected light due to each of the diffraction grating
surfaces, and quantitatively and/or qualitatively analyzes each sample flowing through
the respective flow channel, based on the groove pitch identified for each of the
flow channels.
[0103] A fifth analysis method calculates a groove pitch at which resonance occurs and then
quantitatively and/or qualitatively analyzes a sample based on the obtained groove
pitch. The method employs a surface plasmon resonance sensor chip formed as follows.
On the sensor chip, diffraction grating surfaces are arranged in a direction perpendicular
to the groove orientation, and the sensor chip further comprises a cover for covering
the sensor surface and flow channels formed side by side between the sensor surface
and the cover so as to pass along the direction in which said diffraction grating
surfaces are arranged. Along each of the flow channels, each of the diffraction grating
surfaces has a reaction area, within which the binding substance is immobilized, and
a non-reaction area, within which a substance that does not bind specifically to any
target species in the sample is immobilized or, alternatively, any substance is not
immobilized. This method comprises the steps of: assigning different samples one to
each of the flow channels, and letting each of the samples flow through the respective
flow channel while irradiating the sensor surface with light at a predetermined incident
angle; receiving light reflected from the sensor surface and measuring the intensity
of the light reflected by each of the diffraction grating surfaces; identifying, for
each of the flow channels and for each of the reaction areas and the non-reaction
areas, a groove pitch at which the resonance phenomenon of the evanescent wave and
the surface plasmon wave occurs, based on the measured intensity of the reflected
light due to each of the diffraction grating surfaces; and quantitatively and/or qualitatively
analyzing each sample flowing through the respective flow channel, based on the groove
pitch identified for each of the flow channels and for each of the reaction areas
and the non-reaction areas.
[0104] With this analysis method, not only similar effects to those of the first analysis
method but also the following advantages can be attained. More than one target species
is simultaneously analyzed, thus enabling effective analysis. In addition, since more
than one type of target species can be analyzed under an identical condition, it is
possible to make the difference among the target species clear. Moreover, it is possible
to accurately analyze changes in target species caused by reactions for each of the
flow channels separately.
[0105] This analysis method is realized by an analysis apparatus constructed as follows.
The apparatus comprises: a holding means for holding such a second surface plasmon
resonance sensor chip; a sample introducing means for assigning different samples
one to each of the flow channels, and for introducing each sample into the respective
flow channel in a state where the surface plasmon resonance sensor chip is held by
the holding means; a light irradiating means for irradiating the sensor surface with
light from a predetermined direction in a state where each sample is introduced into
the respective flow channel by the sample introducing means; a light receiving means
for receiving light reflected from the sensor surface; a measuring means for measuring
the intensity of the light reflected by each of the diffraction grating surface and
received by the light receiving means. The apparatus further comprises an analyzing
means for analyzing the sample based on the light received by the light receiving
means. The analyzing means identifies, for each of the flow channels and for each
of the reaction areas and the non-reaction areas, a groove pitch at which a resonance
phenomenon of the evanescent wave and the surface plasmon wave occurs, based on the
intensity, measured by the measuring means, of the reflected light due to each of
the diffraction grating surfaces, and quantitatively and/or qualitatively analyzes
each sample flowing through the respective flow channel, based on the groove pitches
of the reaction area and the non-reaction area identified for each of the flow channels.
[0106] A sixth analysis method calculates the variation in the intensity of reflected light
and then analyzes a sample based on the variation amount. The method comprises the
steps of: making a sample in contact with the sensor surface while irradiating the
sensor surface with light at a predetermined incident angle; receiving the light reflected
from the sensor surface and measuring the intensity of the light reflected by each
of the diffraction grating surfaces; determining the variation between the measured
intensity of the reflected light due to each of the diffraction grating surfaces and
the intensity of the light reflected when any sample is not in contact with the sensor
surface; and selecting a diffraction grating surface whose determined variation of
the reflected light intensity is within a predetermined allowable range (determination
range) for determination, and quantitatively and/or qualitatively analyzing the sample
based on the variation of the reflected light intensity of the selected diffraction
grating surface.
[0107] With this method, even when a wide range of sample concentrations are used, it is
not required to readjust the optical system in such a manner that measurement results
fall within a measurement range, the measurement range thereby being virtually enlarged.
In this case, also, the above steps can be performed in the order of the foregoing
description, or alternatively, they can be performed simultaneously.
[0108] The above-described analysis method is realized by an analysis apparatus constructed
as follows. The apparatus comprises: a holding means for holding such a second surface
plasmon resonance sensor chip with the sensor surface being in contact with a sample;
a light irradiating means for irradiating the sensor surface with light from a predetermined
direction in a state where the surface plasmon resonance sensor chip is held by the
holding means; a light receiving means for receiving light reflected from the sensor
surface; a measuring means for measuring the intensity of the light reflected by each
of the diffraction grating surfaces and received by the light receiving means. The
apparatus further includes a determining means and an analyzing means for analyzing
the sample based on the reflected light received by the light receiving means. The
determining means determines the variation between the intensity, measured by the
measuring means, of the reflected light due to each of the diffraction grating surfaces
and the intensity of the light reflected when any sample is not in contact with the
sensor surface; and the analyzing means selects a diffraction grating surface whose
variation, determined by the determining means, of the reflected light intensity is
within a predetermined allowable range for determination, and quantitatively and/or
qualitatively analyzes the sample based on the variation of the reflected light intensity
of the selected diffraction grating surface.
[0109] A seventh analysis method measures the variation in the intensity of reflected light
and then analyzes a sample based on the variation amount. The method employs a surface
plasmon resonance sensor chip comprising non-diffraction surfaces with no diffraction
grating formed thereon, each of which non-diffraction surfaces is disposed along the
sensor surface on the same plane with the respective one of the diffraction grating
surfaces. This method comprises the steps of: making a sample in contact with the
sensor surface while irradiating the sensor surface with light at a predetermined
incident angle; receiving light reflected from each of the diffraction grating surfaces
and measuring the intensity of the light reflected by each of the diffraction grating
surfaces; correcting the measured intensity of the reflected light due to each of
the diffraction grating surfaces with consideration given to the intensity of the
light reflected by the respective non-diffraction surface; determining the variation
between the corrected intensity of the reflected light due to each of the diffraction
grating surfaces and the intensity of the light reflected when any sample is not in
contact with the sensor surface; and selecting a diffraction grating surface whose
determined variation of the reflected light intensity is within a predetermined allowable
range (determination range) for determination, and quantitatively and/or qualitatively
analyzing the sample based on the variation of the reflected light intensity of the
selected diffraction grating surface.
[0110] With this method, not only similar effects to those of the sixth analysis method
but also the following advantages can be attained. Since the intensity of light reflected
from each diffraction grating surface is corrected in consideration of the intensity
of light reflected from a non-diffraction surface, it is possible to correct errors
in the intensity of reflected light caused by differences in surface properties among
the diffraction grating surfaces.
[0111] This analysis method is realized by an analysis apparatus constructed as follows.
The apparatus comprises: a holding means for holding such a second surface plasmon
resonance sensor chip with the sensor surface being in contact with a sample; a light
irradiating means for irradiating the sensor surface with light from a predetermined
direction in a state where the surface plasmon resonance sensor chip is held by the
holding means; a light receiving means for receiving light reflected from the sensor
surface; and a measuring means for measuring the intensity of the light reflected
by each of the diffraction grating surfaces and received by the light receiving means.
The apparatus further comprises a correcting means, a determining means, and an analyzing
means, for analyzing the sample based on the reflected light received by the light
receiving means. The correcting means corrects the intensity of the reflected light
due to each of the diffraction grating surfaces with consideration given to the intensity
of the reflected light due to the respective non-diffraction surface; the determining
means determines the variation between the intensity, corrected by the correcting
means, of the reflected light due to each of the diffraction grating surfaces and
the intensity of the light reflected when any sample is not in contact with the sensor
surface; and the analyzing means selects a diffraction grating surface whose variation,
determined by the determining means, of the reflected light intensity is within a
predetermined allowable range for determination, and quantitatively and/or qualitatively
analyzes the sample based on the variation of the reflected light intensity of the
selected diffraction grating surface.
[0112] An eighth analysis method measures the variation in the intensity of reflected light
and then analyzes a sample based on the variation amount. The method employs a surface
plasmon resonance sensor chip formed as follows: each of the diffraction grating surfaces
has a reaction area, within which the binding substance is immobilized, and a non-reaction
area, within which a substance that does not bind specifically to any target species
in the sample is immobilized or, alternatively, any substance is not immobilized.
This analysis method comprises the steps of: making a sample in contact with the sensor
surface while irradiating the sensor surface with light at a predetermined incident
angle; receiving light reflected from each of the diffraction grating surfaces and
measuring the intensity of the light reflected by each of the diffraction grating
surfaces; determining, for each of the reaction areas and the non-reaction areas,
the variation between the measured intensity of the reflected light due to each of
the diffraction grating surfaces and the intensity of the light reflected when any
sample is not in contact with the sensor surface; selecting, for each of the reaction
areas and the non-reaction areas, a diffraction grating surface whose determined variation
of the reflected light intensity is within a predetermined allowable range for determination,
and quantitatively and/or qualitatively analyzing the sample based on the variation
of the reflected light intensity of the selected reaction area and the variation of
the reflected light intensity of the selected non-reaction area.
[0113] With this method, not only similar effects to those of the sixth analysis method
but also the following advantages can be attained. Since the analysis is made based
on a groove pitch in a reaction area and that in a non-reaction area, it is possible
to accurately analyze changes in target species caused by reactions.
[0114] This analysis method is realized by an analysis apparatus constructed as follows.
The apparatus comprises: a holding means for holding such a second surface plasmon
resonance sensor chip with the sensor surface being in contact with the sample; a
light irradiating means for irradiating the sensor surface with light from a predetermined
direction in a state where the surface plasmon resonance sensor chip is held by the
holding means; a light receiving means for receiving light reflected from each of
the diffraction grating surfaces; and a measuring means for measuring the intensity
of the light reflected by each of the diffraction grating surfaces and received by
the light receiving means. The apparatus further comprises a determining means and
an analyzing means, for analyzing the sample based on the reflected light received
by the light receiving means. The determining means determines, for each of the reaction
areas and the non-reaction areas, the variation between the intensity, measured by
the measuring means, of the reflected light due to each of the diffraction grating
surfaces and the intensity of the light reflected when any sample is not in contact
with the sensor surface; and the analyzing means selects, for each of the reaction
areas and the non-reaction areas, a diffraction grating surface whose determined variation
of the reflected light intensity is within a predetermined allowable range for determination,
and for quantitatively and/or qualitatively analyzes the sample based on the variation
of the reflected light intensity of the selected reaction area and the variation of
the reflected light intensity of the selected non-reaction area.
[0115] A ninth analysis method measures the variation in the intensity of reflected light
and then analyzes a sample based on the variation amount. The method employs a surface
plasmon resonance sensor chip formed as follows on the sensor chip: such diffraction
grating surfaces are arranged in a direction perpendicular to the groove orientation,
and the sensor chip further comprises a cover for covering the sensor surface, and
flow channels formed side by side between the sensor surface and the cover so as to
pass along the direction in which the diffraction grating surfaces are arranged. This
analysis method comprises the steps of: assigning different samples one to each of
the flow channels, and letting each of the samples flow through the respective flow
channel while irradiating the sensor surface with light at a predetermined incident
angle; receiving light reflected from the sensor surface and measuring the intensity
of the light reflected by each of the diffraction grating surfaces; determining the
variation between the measured intensity of the reflected light due to each of the
diffraction grating surfaces and the intensity of the light reflected when any sample
does not flow through each of the flow channel; selecting, for each of the flow channels,
a diffraction grating surface whose determined variation of the reflected light intensity
is within a predetermined allowable range for determination, and quantitatively and/or
qualitatively analyzing each sample flowing through the respective flow channel, based
on the variation of the reflected light intensity of the selected diffraction grating
surface for each of the flow channels.
[0116] With this method, not only similar effects to those of the sixth analysis method
but also the following advantages can be attained. Different kinds of samples are
analyzed simultaneously, thus realizing effective analysis. In addition, since the
different samples are analyzed under an identical condition, it is possible to make
the difference among the samples clear.
[0117] This analysis method is realized by an analysis apparatus constructed as follows.
The apparatus comprises: a holding means for holding such a second surface plasmon
resonance sensor chip; a sample introducing means for assigning different samples
one to each of the flow channels, and for introducing each of the samples into the
respective flow channel in a state where the surface plasmon resonance sensor chip
is held by the holding means; a light irradiating means for irradiating the sensor
surface with light from a predetermined direction in a state where each sample is
introduced into the respective flow channel by the sample introducing means; a light
receiving means for receiving light reflected from each of the diffraction grating
surfaces; a measuring means for measuring the intensity of the light reflected by
each of the diffraction grating surfaces and received by the light receiving means.
The apparatus further comprises a determining means and an analyzing means, for analyzing
the sample based on the reflected light received by the light receiving means. The
determining means determines the variation between the intensity, measured by the
measuring means, of the reflected light due to each of the diffraction grating surfaces
and the intensity of the light reflected when any sample does not flow through the
flow channels; and the analyzing means selects, for each of the flow channels, a diffraction
grating surface whose variation, determined by the determining means, of the reflected
light intensity is within a predetermined allowable range for determination, and quantitatively
and/or qualitatively analyzes each sample flowing through the respective flow channel
based on the variation of the reflected light intensity of the diffraction grating
surface selected for each of the flow channels.
[0118] A 10th analysis method measures the variation in the intensity of reflected light
and then analyzes a sample based on the variation amount. The method employs a surface
plasmon resonance sensor chip formed as follows. On the sensor chip, diffraction grating
surfaces are arranged in a direction perpendicular to the groove orientation. The
sensor chip further comprises: a cover for covering the sensor surface; and flow channels
formed side by side between the sensor surface and the cover so as to pass along the
direction in which the diffraction grating surfaces are arranged. Along each of the
flow channels, each of the diffraction grating surfaces has a reaction area, within
which the binding substance is immobilized, and a non-reaction area, within which
a substance that does not bind specifically to any target species in the sample is
immobilized or, alternatively, any substance is not immobilized. This analysis method
comprises the steps of: assigning different samples one to each of the flow channels,
and letting each of the samples flow through the respective flow channel while irradiating
the sensor surface with light at a predetermined incident angle; receiving the light
reflected from the sensor surface and measuring the intensity of the light reflected
by each of the diffraction grating surface; determining, for each of the reaction
area and the non-reaction area, the variation between the measured intensity of the
reflected light due to each of the diffraction grating surfaces and the intensity
of the light reflected when any sample does not flow through the flow channels; selecting,
for each of the flow channels and for each of the reaction areas and the non-reaction
areas, a diffraction grating surface whose determined variation of the reflected light
intensity is within a predetermined allowable range for determination, and quantitatively
and/or qualitatively analyzing each sample flowing through the respective flow channel,
based on the variation of the reflected light intensity of the selected reaction area
and the variation of the reflected light intensity of the selected non-reaction area.
[0119] With this method, not only similar effects to those of the sixth analysis method
but also the following advantages can be attained. Different kinds of samples are
analyzed simultaneously, thus realizing effective analysis. In addition, since the
different samples are analyzed under an identical condition, it is possible to make
the difference among the samples clear. Furthermore, since the analysis is performed
based on the groove pitch of a reaction area and that of a non-reaction area, it is
possible to accurately analyze changes in target species caused by reactions for each
of the flow channels separately.
[0120] This method is realized by an analysis apparatus constructed as follows. The apparatus
comprises: a holding means for holding such a second surface plasmon resonance sensor
chip; a sample introducing means for assigning different samples one to each of the
flow channels, and for introducing each of the plural samples into the respective
flow channel in a state where the surface plasmon resonance sensor chip is held by
the holding means; a light irradiating means for irradiating the sensor surface with
light from a predetermined direction in a state where each sample is introduced into
the respective flow channel by the sample introducing means; a light receiving means
for receiving light reflected from the sensor surface; and a measuring means for measuring
the intensity of the light reflected by each of the diffraction grating surfaces and
received by the light receiving means. The apparatus further comprises a determining
means and an analyzing means, for analyzing the sample based on the light received
by the light receiving means. The determining means determines, for each of the reaction
areas and the non-reaction areas, the variation between the intensity, measured by
the measuring means, of the reflected light due to each of the diffraction grating
surfaces and the intensity of the light reflected when any sample does not flow through
the flow channels; and the analyzing means selects, for each of the flow channels
and for each of the reaction areas and the non-reaction areas, a diffraction grating
surface whose variation, determined by the determining means, of the reflected light
intensity is within a predetermined allowable range for determination, and quantitatively
and/or qualitatively analyzes each sample flowing through the respective flow channel,
based on the variation of the reflected light intensity of the selected reaction area
and the variation of the reflected light intensity of the selected non-reaction area
for each of the flow channels.
[0121] As a preferred feature, each of the foregoing methods further comprises the step
of separating the sample by physical and/or chemical action prior to introducing the
sample to the surface plasmon resonance sensor chip.
[0122] With this feature, it is possible to appropriately remove impurities, if any, other
than target species contained in the sample before analysis, so that only pure target
species can be subject to the analysis. As a consequence, the accuracy of the analysis
is improved.
[0123] At that time, if combined with detecting techniques (absorbance detection, fluorescence
detection, chemiluminescence detection, differential refract meter detection, electrochemical
detection, etc.) that are commonly used in these analyzing means, it is possible to
perform the following determination at the same time: determination of the presence
amount of various substances; and determination of target species by measuring reactions
unique to the target species.
[0124] This analysis method is implemented by an analysis apparatus which has, in addition
to the construction as described above, a sample separating means for separating the
sample by physical and/or chemical action prior to introducing the sample to the surface
plasmon resonance sensor chip.
[0125] The following separation techniques preferably serve as such sample separating means:
liquid chromatography; HPLC (High Performance Liquid Chromatography); capillary electrophoresis;
microchip electrophoresis; and methods using flow injection or microchannels.
[0126] As a preferred feature, if the target species is a light-emitting substance, the
method further comprises the step of detecting light emitted from the light-emitting
substance that is bound to the binding substance prior to light-irradiating the sensor
surface or, alternatively, after light-irradiating the sensor surface and receiving
the reflected light. In the step of quantitatively and/or qualitatively analyzing,
the sample is analyzed with consideration given to the detection result of the light
emission.
[0127] With this feature, light-emitting phenomena as well as surface plasmon resonance
can be utilized in the analysis. The extremely high sensitivity of light-emitting
phenomena, such as fluorescence and phosphor, enables detection of minute reactions.
[0128] This analyzing means can be implemented by an analysis apparatus that has, in addition
to the construction as described above, the following construction: when the target
species is a light-emitting substance, the light receiving means detects light emitted
from the light-emitting substance that is bound to the binding substance, and the
analyzing means quantitatively and/or qualitatively analyzes the sample with consideration
given to the detection result of the light emission by the light receiving means.
[0129] In addition to the foregoing first and second surface plasmon resonance sensor chips,
another type of surface plasmon resonance sensor chip with the following construction
realizes diffraction gratings having distributed groove pitches when viewed from the
incident direction of the incident light. This surface plasmon resonance sensor chip
(a third sensor chip) comprises: a metal layer along whose surface a surface plasmon
wave can be induced by light irradiation; and a diffraction grating curved surface
disposed in the vicinity of the metal layer, which diffraction grating curved surface
has a diffraction grating with a uniform groove orientation and a uniform groove pitch
so as to generate an evanescent wave upon light irradiation. The diffraction grating
curved surface has a curved surface form in a convex shape whose light-irradiated
side bulges out, and is disposed so as to be perpendicular to a specific plane, which
is perpendicular to a predetermined reference plane, and the diffraction grating is
formed in such a manner that the groove orientation is perpendicular to the specific
plane.
[0130] With the thus-constructed sensor chip, when a light beam (parallel light) is emitted
from a predetermined direction in parallel to the above-mentioned specific plane,
the irradiated light enters onto the diffraction grating surface at distributed angles,
depending on inclination angles which are formed between tangent planes at different
irradiated positions and the reference plane. The intensity of reflected light obtained
at different positions on the diffraction grating surface also reveals distribution.
Therefore, it is possible to calculate a resonance angle in real time based on the
intensity of reflected light at each position on the diffraction grating surface and
a substantial incident angle at the position. In other words, with such a sensor chip,
similar effects to those that are attained when a light beam having a predetermined
spread angle (to widen or narrow the light) is used are accomplished, without necessity
to perform angle scanning or to irradiate a wedge-shaped light beam.
[0131] As a preferred feature, the diffraction grating curved surface has a curved surface
form in a convex shape whose light-irradiated side bulges out. With this feature,
it is possible to prevent crossing of the light reflected from different positions
on the diffraction grating surface, thus facilitating analysis of the intensity of
light reflected from the diffraction grating surfaces.
[0132] As another preferred feature, two or more of such diffraction grating surfaces are
prepared. In this case, it is possible to detect resonance angles of the separate
diffraction grating surfaces at the same time, by simply irradiating a light beam
from a predetermined direction in parallel to the specific plane. Therefore, with
use of a binding substance immobilized according to each of the diffraction grating
surfaces, simultaneous measurement at different positions is available.
[0133] On the above-described third sensor chip, each of the diffraction grating surfaces
is disposed along a sensor surface, which comes in contact with a sample. When the
third sensor chip is used to quantitatively and/or qualitatively analyze a sample,
immobilized binding substances that bind specifically to target species in the sample
are employed on the sensor surface. In particular, in a case where a sensor chip for
multiple-position simultaneous measurement is used, two or more binding substances
each of which binds to a specific target species in the sample are immobilized in
association with each of the diffraction grating surfaces, thus making it possible
to assess two or more target species simultaneously.
[0134] Next, another type of surface plasmon resonance sensor chip (a fourth sensor chip)
has a resonance area, formed on its sensor surface, for causing a resonance phenomenon
of a surface plasmon wave, which is induced along the surface of the metal layer,
and an evanescent wave, which is generated by the action of the diffraction grating,
upon light irradiation. The resonance area has continuous areas discretely formed
on the sensor surface, and at least one of the continuous areas has a diffraction
grating whose groove pitch or/and whose groove orientation is different from those
of the remaining continuous areas. Here, the above-mentioned continuous areas are
defined as a set of areas that are considered to be continuously arranged on the same
plane from the viewpoint of whether or not a resonance phenomenon occurs.
[0135] With this construction, when viewed from a predetermined direction the diffraction
grating has substantially distributed groove pitches, and in each of the continuous
areas, an evanescent wave is induced that has the number of waves and an angular frequency
corresponding to the substantial groove pitch. As a result, resonance phenomena occur
at two or more resonance points that reveal different angular frequencies, for one
single surface plasmon wave.
[0136] Further, still another type of surface plasmon resonance sensor chip (a fifth sensor
chip) has resonance areas continuously formed on its sensor surface, in which area
a resonance phenomenon occurs between a surface plasmon wave, which is induced along
the surface of the metal layer, and an evanescent wave, which is generated by the
action of the diffraction grating, upon light irradiation. The resonance areas are
continuously formed on the sensor surface, and in the diffraction gratings the grooves
are oriented along a uniform direction at groove pitches with a continuous or discontinuous
distribution. Here, the above-mentioned "continuous distribution" means a condition
where every groove is spaced from its adjacent one at a slightly different distance
so that the distance between every pair of grooves adjacent to each other gradually
increases or decreases in a certain direction. Meanwhile, the above-mentioned "discontinuous
distribution" means a condition where the inter-groove distance is changed in a step-like
form between at least one pair of adjacent grooves.
[0137] With this sensor chip, it is possible to obtain an evanescent wave with the number
of waves and an angular frequency corresponding to the groove pitch at each position
on the continuous areas. As a result, resonance phenomena occur at two or more resonance
points with different angular frequencies for one single surface plasmon wave.
[0138] When a foregoing fourth or fifth sensor chip is used to quantitatively and/or qualitatively
assess a sample, immobilized binding substances (substances which can capture target
species as a result of interaction such as antigen-antibody reaction, complementary
DNA bonding, receptor-ligand interaction, enzyme-substrate interaction) which binds
specifically to target species (chemical species, biochemical species, or biological
species, etc.) in the sample are employed on the sensor surface. Then the sample is
analyzed in accordance with the analysis method comprising a light-irradiating step,
a detecting step, and an analyzing step, as described below.
[0139] The light-irradiating step makes the sample in contact with a sensor surface while
irradiating the sensor surface with light from a predetermined direction. The detecting
step receives light reflected from a resonance area and detects a resonance phenomenon
in the resonance area based on the intensity of the thus-received reflected light.
The analyzing step quantitatively and/or qualitatively analyzes the sample based on
the substantial groove pitch of the diffraction grating, viewed from the incident
light direction at a position where a resonance phenomenon is detected. Here, the
above steps can be performed in the order of description, or alternatively, they can
be performed simultaneously. In the latter case, particularly, it is possible to monitor
in real time how target species bind to binding substances.
[0140] The above analysis method is realized by an analysis apparatus constructed as follows.
The apparatus comprises: a holding means for holding the surface plasmon resonance
sensor chip with the sensor surface being in contact with a sample; a light irradiating
means for emitting a light beam toward a resonance area of the sensor chip in a state
where the surface plasmon resonance sensor chip is held by the holding means; and
a light receiving means for receiving light reflected from the resonance area.
[0141] The apparatus further includes a detecting means and an analyzing means, for analyzing
the sample based on the reflected light received by the light receiving means. The
detecting means detects a resonance phenomenon in the resonance area based on the
intensity of the thus-received reflected light. The analyzing step quantitatively
and/or qualitatively analyzes the sample based on the substantial groove pitch of
the diffraction grating, viewed from the incident light direction at a position where
a resonance phenomenon is detected.
[0142] As a preferred feature, in the above analyzing step, the quantitative and/or qualitative
analysis of the sample is performed based on the substantial groove pitch of the diffraction
grating, viewed from the incident light direction at a position where a resonance
phenomenon is detected, and also on the intensity of reflected light detected at the
position where a resonance phenomenon is detected. In this case, the analysis apparatus
needs to be provided with a analyzing means for quantitatively and/or qualitatively
analyzing the sample based on the substantial groove pitch in the diffraction grating,
viewed from the incident light direction at a position where a resonance phenomenon
is detected, and also on the intensity of reflected light detected at the position
where a resonance phenomenon is detected.
[0143] Further, as another preferred feature, in the above irradiating step, a light beam
of a single wavelength can be emitted at a predetermined incident angle. In this case,
the analysis apparatus needs to have a light irradiating means for emitting a light
beam of a single wavelength at a predetermined incident angle. Using the foregoing
surface plasmon sensor chip, it is still possible to detect a resonance point, where
a resonance phenomenon occurs, with such a simple optical system. At this time, it
is particularly preferred to use a surface plasmon resonance chip with a continuous
groove pitch distribution.
Brief Description of the Drawings
[0144]
FIG. 1 is an oblique perspective diagram showing the constitution of a sensor chip
according to the first embodiment of the present invention;
FIG. 2 is an oblique perspective diagram showing the constitution of a substantial
part of the sensor chip of FIG. 1;
FIG. 3 is an oblique perspective diagram illustrating the state where a binding substance
is immobilized on the sensor chip of FIG. 1;
FIG. 4 is an oblique perspective diagram illustrating an example of a method for making
the sensor chip of FIG. 1, (a)-(c) each indicating making processes in sequence;
FIG. 5 is a diagram showing the constitution of an analysis apparatus according to
the first embodiment of the present invention;
FIG. 6 is an oblique perspective diagram showing the constitution of a substantial
part of a sensor chip according to the second embodiment of the present invention;
FIG. 7 is an oblique perspective diagram showing the constitution of a substantial
part of a sensor chip according to the third embodiment of the present invention;
FIG. 8 is an oblique perspective diagram illustrating the state where a binding substance
and a non-binding substance are immobilized on a sensor chip according to the fourth
embodiment of the present invention;
FIG. 9 is a diagram showing the constitution of an analysis apparatus according to
the fifth embodiment of the present invention;
FIG. 10 is an oblique perspective diagram showing the constitution of a substantial
part of a sensor chip according to the seventh embodiment of the present invention;
FIG. 11 is a diagram showing the constitution of an analysis apparatus according to
the seventh and tenth embodiments of the present invention;
FIG. 12 is an oblique perspective diagram showing the constitution of a sensor chip
according to the eighth embodiment of the present invention;
FIG. 13 is an oblique perspective diagram showing the constitution of a sensor chip
according to the ninth embodiment of the present invention;
FIG. 14 is an oblique perspective diagram showing the constitution of a sensor chip
according to the tenth embodiment of the present invention;
FIG. 15 is an oblique perspective diagram showing a sensor chip according to the eleventh
embodiment of the present invention;
FIG. 16 is an oblique perspective diagram illustrating the state where a binding substance
is immobilized on the sensor chip of FIG. 15;
FIG. 17 is a dispersion relationship diagram showing optical properties of the sensor
chip of FIG. 15;
FIG. 18 is an oblique perspective diagram illustrating an example of a method for
making the sensor chip of FIG. 15, (a)-(c) each indicating making processes in sequence;
FIG. 19 is an oblique perspective diagram showing a modification of the sensor chip
of FIG. 15;
FIG. 20 is a diagram showing an analysis apparatus according to an embodiment of the
present invention;
FIG. 21(a) is an oblique perspective diagram showing a sensor chip according to the
twelfth embodiment of the present invention, and FIG. 21(b) is a schematic diagram
for illustrating a sensor chip of the present invention;
FIG. 22 is an oblique perspective diagram showing a sensor chip according to the thirteenth
embodiment of the present invention;
FIG. 23 is a diagram showing an analysis apparatus according to the fourteenth embodiment
of the present invention;
FIG. 24 is an oblique perspective diagram showing a sensor chip according to the fifteenth
embodiment of the present invention;
FIG. 25 is a diagram showing an analysis apparatus according to the fifteenth embodiment
of the present invention;
FIG. 26(a) and FIG. 26(b) are elevational views each illustrating a sensor chip according
to the sixteenth embodiment of the present invention;
FIG. 27(a), FIG. 27(b), and FIG. 27(c) are schematic diagrams each for illustrating
a sensor chip according to the sixteenth embodiment of the present invention;
FIG. 28(a), FIG. 28(b), and FIG. 28(c) are plane views each showing a sensor chip
according to the seventeenth embodiment of the present invention;
FIG. 29 is an oblique perspective diagram each showing a sensor chip according to
the eighteenth embodiment of the present invention;
FIG. 30 is an oblique perspective diagram showing a sensor chip according to the nineteenth
embodiment of the present invention;
FIG. 31 is a diagram showing the distribution of groove pitches on the sensor chip
of FIG. 30;
FIG. 32 is a dispersion relationship diagram showing optical properties of the sensor
chip of FIG. 30;
FIG. 33 is a plane view showing a sensor chip according to the twentieth embodiment
of the present invention;
FIG. 34 is an oblique perspective diagram showing a modification of the arrangement
pattern of diffraction grating surfaces according to the sensor chip of FIG. 1;
FIG. 35 is an oblique perspective diagram showing another modification of the arrangement
pattern of diffraction grating surfaces according to the sensor chip of FIG. 1;
FIG. 36 is an oblique perspective diagram showing another modification of arrangement
pattern of diffraction grating surfaces according to the sensor chip of FIG. 1;
FIG. 37 is an oblique perspective diagram showing a modification of the sensor chip
shown in FIG. 19;
FIG. 38 is an oblique perspective diagram showing another embodiment of a sensor chip
of the present invention;
FIG. 39 is an oblique perspective diagram showing a sensor chip according to the first
example of the present invention;
FIG. 40 is an oblique perspective diagram showing another sensor chip according to
the first example of the present invention;
FIG. 41 is a graph showing the incident angle of incident light and reflected light
intensity according to the first example of the present invention;
FIG. 42 is another graph showing the incident angle of incident light and reflected
light intensity according to the first example of the present invention;
FIG. 43 is a graph showing the ethanol concentration of ethanol aqueous solution and
the shift amount of resonance angle according to the third example of the present
invention;
FIG. 44 is another graph showing the ethanol concentration of ethanol aqueous solution
and the shift amount of resonance angle according to the third example of the present
invention;
FIG. 45 is a graph showing the ethanol concentration of ethanol aqueous solution and
reflected light intensity according to the fourth example of the present invention;
FIG. 46 is another graph indicating the ethanol concentration of ethanol aqueous solution
and reflected light intensity according to the fourth example of the present invention;
FIG. 47 is an oblique perspective diagram showing a sensor chip according to the fifth
example of the present invention;
FIG. 48 is a graph showing the results of the first example;
FIG. 49 is another graph showing the results of the first example;
FIG. 50(a) and FIG. 50(b) are graphs each showing the results of the second example;
FIG. 51 is a dispersion relationship diagram showing properties of the conventional
sensor chip; and
FIG. 52(a) is a diagram showing the optical system of the conventional angle-varying
type analysis apparatus, and FIG. 52(b) is a magnified diagram showing a light-irradiated
state of a sensor chip.
Best Mode for Carrying Out the Invention
[0145] In the following, embodiments of the present invention will be described with reference
to the figures.
(A) First Embodiment
[0146] At first, the first embodiment of the present invention is described with reference
to FIGs. 1-5.
[0147] As shown in FIG. 1, the sensor chip according to the present embodiment (surface
plasmon resonance sensor chip) 1 is laminated with a metal layer 3 on its surface
(sensor surface) 1a, and partly on the metal layer 3, plural diffraction areas 6 are
disposed in plural positions, respectively. A diffraction grating is formed within
each of the diffraction areas 6. In the present embodiment, each diffraction area
6 within which a diffraction grating is formed is used as a measurement spot during
analysis by multipoint simultaneous measurement.
[0148] FIG. 2 is an oblique perspective diagram showing a measurement spot 6 under magnification.
As shown in FIG. 2, plural planes (hereinafter called diffraction grating surfaces)
5a-5i are concentratedly disposed in the measurement spot 6, and a diffraction grating
is formed within each of the diffraction grating surfaces. If the remaining surface
of the metal layer 3 other than the measurement spots 6 is assumed as a reference
plane S0, each of the diffraction grating surfaces 5a-5i is disposed so as to be perpendicular
to a specific plane S1, which is perpendicular to the reference plane S0, and so as
to form a predetermined inclination angle αa-αi with the reference plane S0. In the
diffraction grating surfaces 5a-5i, diffraction gratings having the same shape (the
same groove depth, the same groove pitch) are formed in such a manner that the groove
orientation of the diffraction gratings is perpendicular to the specific plane S1.
[0149] In this embodiment, the central diffraction grating surface 5e is disposed in parallel
with the reference plane S0, while the remaining diffraction grating surfaces 5a-5d,
5f-5i are arranged in such a manner that the inclination angles that these diffraction
grating surfaces 5a-5d, 5f-5i form with the reference plane S0 gradually increase
with distant from the center. Put it another way, when viewed from a direction A parallel
to the specific plane, the diffraction grating surfaces 5a-5d, 5f-5i are positioned
in decreasing order of the inclination angles that the diffraction grating surfaces
5a-5d, 5f-5i form with the reference plane S0 (i.e., αa > αb > αc > αd > αe (αe=0)
> αf > αg > αh > αi). The diffraction grating surfaces 5a-5i are also arranged in
such a manner that two adjacent diffraction grating surfaces are positioned continuously.
[0150] With this structure, when the sensor surface 1a of the sensor chip 1 is irradiated
with light, the irradiation light is diffracted at each of the measurement spots 6
on the sensor surface 1a, and this diffraction phenomenon generates an evanescent
wave. In this case, the substantial incident angles at each measurement spot 6 differ
among the diffraction grating surfaces 5a-5i. In FIG. 2, when the incident angle that
the irradiation light forms with the reference plane S0 is assumed as θ, actual incident
angles on the diffraction grating surfaces 5a-5i are represented in turn, from the
diffraction grating surface 5a positioned at the end of the light-irradiated side,
as θ-αa, θ-αb, θ-αc, ···, θ-αi. Since the incident angles of the irradiation light
thus differ among the diffraction grating surfaces 5a-5i, the wave number of the evanescent
wave generated by diffraction phenomenon becomes different among the diffraction grating
surfaces 5a-5i. Further, when the irradiation light affects the metal layer 3 to thereby
produce resonance (SPR) with a surface plasmon wave, the degree of the resonance (SPR)
along the surface of the metal layer 3 also differs accordingly among the diffraction
grating surfaces 5a-5i.
[0151] It is possible to make the sensor chip 1 by the following processes. Using a substrate
2 shown in FIG. 4(a), the first process is, as shown in FIG. 4(b), to form plural
uneven surfaces 4 each of which has uneven form in plural positions, respectively,
partly on the substrate surface by laser processing or the like. The second process
is, as shown in FIG. 4(c), to laminate all over the surface of the substrate 2 with
a metal layer 3 by sputtering, vapor deposition, or the like. Since the metal layer
3 is layered on the uneven surfaces 4, the surface of the metal layer 3 also has uneven
surfaces thereon. These uneven surfaces on the the metal layer 3 surface function
as diffraction grating surfaces, and each of diffraction areas having the uneven surfaces
4 formed therein are used as a measurement spot 6.
[0152] The material used for the substrate 2 is not limited as long as it allows the uneven
surfaces 4 to be formed on its surface and as it possesses adequate mechanical strength
to hold the metal layer 3. Resins are desirable in the point that the uneven surfaces
4 are easily formable. Preferable materials include acrylic resin (such as poly(methyl
methacrylate)), polyester resin (such as polycarbonate), and polyolefin.
[0153] The material used for the metal layer 3 is not limited as long as it can induce a
surface plasmon wave. Examples of the usable material include gold, silver, copper,
aluminum, alloies containing one or more of the preceding metals, and oxides of silver,
copper, and aluminum. Among above, silver is preferable in respect of sensitivity
and inexpensiveness, while gold is preferable in point of stability. The thickness
of the metal layer 3 is preferably between 20-300 nm, more preferably between 30-160
nm. If the thickness of the metal layer 3 is too small, the irradiation light passes
through the metal layer 3 and reaches the surface of the substrate 2, and may be diffracted
by the uneven surfaces 4 on the surface of the substrate 2. In this case, the uneven
forms of the uneven surfaces 4 may also function as diffraction gratings.
[0154] It is possible to form the uneven surfaces 4 on the substrate 2 not only by laser
processing as mentioned above, but also by injection-molding into a metal mold having
a predetermined uneven form, which mold can be made by an ion beam, to integrally
mold uneven surfaces 4 with the substrate 2. Alternatively, it is also possible to
form desired uneven surfaces 4 by forming a plane, which have an angle of inclination
but does not have uneven form, on the surface of the substrate 2 in advance, and then
pasting a permeable uneven film on the plane. In addition, it is also possible to
form desired uneven surfaces 4 by processing the plane using microcutting technique
or by microcontact-printing PDMS (poly(dimethylsiloxane)) on the plane to thereby
form uneven form having projections and depressions.
[0155] When making the uneven form of the uneven surfaces 4, it is required to consider
the thickness of the metal layer 3 or the likes in such a manner that when the metal
layer 3 is layered, a desired diffraction grating is obtained on the surface. The
uneven form can be a square-wave form, a sine-wave form, a saw-tooth form, etc., preferably
a cyclic uneven form whose diffraction grating has a groove depth (from the top to
the bottom) of between 10-200nm (more preferably, between 30-120nm) and a groove pitch
(a cycle: a distance from a protrusion to an adjacent protrusion in the uneven form)
of between 400-1200nm (which value relates to the wavelength of the irradiation light).
[0156] The number of diffraction grating surfaces formed in each measurement spot 6 is within
the range from 2 to 100, preferably within the range from 5 to 50. The inclination
angle each diffraction grating surface forms with the reference plane is within the
range from -10 to 10 degrees, preferably within the range from -5 to 5 degrees, more
preferably within the range from -3 to 3 degrees. The variation of the inclination
angle between each two adjacent diffraction grating surfaces is within the range from
0.001 to 1 degree, preferably within the range from 0.01 to 0.5 degree.
[0157] The size of each diffraction grating surface depends on the number of the measurement
spots 6. When each diffraction grating surface has a rectangular shape, the short
side is within the range from 5µm to 20mm, preferably within the range from 20µm to
5mm. When each diffraction grating surface has a round shape, the diameter is within
the range from 5µm to 20mm, preferably within the range from 20µm to 5mm. The measurement
spots 6 are to be formed in a density of between 0.1-1,000,000 spots/cm
2, preferably between 1-100,000 spots/cm
2. Such a density allows multipoint simultaneous measurement of between 1-10,000,000
measurement spots 6 per single chip.
[0158] Then, a method for using the sensor chip 1 according to the present embodiment is
described.
[0159] When using the sensor chip 1 for sample analysis, the first process is, as shown
in FIG. 3, to immobilize a binding substance 7 on each measurement spot 6. The binding
substance 7 is a substance that has the property of binding specifically to a specific
substance by some interaction such as antigen-antibody reaction, complementarily binding
of DNA, receptor-ligand interaction, or enzyme-substrate interaction. The binding
substance 7 is selected in accordance with a target species to be detected (chemical
species, biochemical species, biological species, etc.). When the sample contains
plural target species, plural binding substance 7 are selected according to the plural
target species, respectively, and immobilized on different measurement spots 6.
[0160] Then, the next process is to set the sensor chip 1, on which the binding substance
7 is immobilized, to an analysis apparatus 10 having the constitution as shown in
FIG. 5, so as to carry out analysis. The analysis apparatus 10 is constituted basically
of a holder 11 for holding the sensor chip 1 fixedly, a light source 12, a light detector
13, and an analysis unit 14.
[0161] The holder 11 has a flow channel 11a formed thereon, through which a sample fluid
containing a target species passes. The sensor chip 1 is disposed and fixed in such
a manner that the sensor surface 1a is in contact with the sample flowing through
the flow channel 11a.
[0162] The light source 12 is disposed opposite to the sensor chip 1 across the flow channel
11a so as to irradiate the sensor surface 1a of the sensor chip 1 with light. The
light source 12 is adjusted such as to light-irradiate in a direction that is parallel
with the specific plane S1 and that forms a predetermined incident angle θ with the
reference plane S0. It is preferable to adjust the incident angle θ in such a manner
that among the individual lights reflected by the diffraction grating surfaces 5a-5i,
the light reflected by the diffraction grating surface 5a with the minimum incident
angle has the minimum intensity. As the light source 12, it is desirable to use a
laser light source that emits monochromatic light, and specifically, a semiconductor
laser in respect of cost and size. The wavelength of the light source 12 is preferably
on the order of 350-1300nm. It is also possible to spectrally separate white light,
which is emitted from a halogen tungsten lamp or others, using interference filter,
spectroscope, or the like to thereby obtain monochromatic light for the light source
purpose.
[0163] The light detector 13 is a detector for detecting the reflection light from the sensor
chip 1, disposed opposite to the sensor chip 1 across the flow channel 11a, as is
the light source 12. As the light detector 13, it is desirable to use, for example,
integrated CCD elements, a silicon photodiode array, etc. In addition, although not
illustrated in the figure, a polarizer is disposed between the light source 12 and
the sensor chip 1 or between the sensor chip 1 and the light detector 13 so as to
polarize the irradiation light from the light source 12 or the reflection light from
the sensor chip 1, since only polarized light P can cause resonance with a surface
plasmon wave.
[0164] The analysis unit 14 is a unit for carrying out analysis processing based on the
detection data from the light detector 13. The analysis unit 14 functions as calculating
means or measuring means and also as analyzing means according to the present invention.
The following description is given to the detailed explanation of the individual functions
of the analysis unit 14, and also to a sample analysis procedure using the sensor
chip 1 according to the present embodiment. The explanation is made individually for
the following two cases: the case where the resonance angle is calculated and the
sample analysis is carried out based on the resonance angle; and the case where the
variation of the reflection-light intensity is measured and the sample analysis is
carried out based on the variation of the reflection-light intensity.
[0165] In the case where the sample analysis is carried out based on the resonance angle,
the first process is to set the sensor chip 1 to the holder 11 and to make the sensor
surface 1a of the sensor chip 1 in contact with the sample (Step A1). The binding
substance 7 imobilized on the individual measurement spot 6 of the sensor surface
1a thereby comes to bind specifically to the target species contained in the sample
fluid. According to the amount of substance of the target species bound to the binding
substance 7, the refractive index of a medium adjacent to the metal layer 3 surface
on each measurement spot 6 changes, and conditions under which the resonance of a
surface plasmon wave occurs at each measurement spot 6 also change accordingly.
[0166] The second process is to irradiate the sensor surface 1a with light from the light
source 12 (step A2). At the time, the area of the irradiation light is adjusted such
that the irradiation light illuminates all the measurement spots 6. The irradiation
light entered to the sensor surface 1a produces diffraction lights on the individual
diffraction grating surfaces 5a-5i disposed in each measurement spot 6. Among these
diffraction lights, the light detector 13 detects the diffraction light of the 0th-order
(reflection light), and measures the intensity of the reflection light detected (step
A3). Thus the light detector functions as both light receiving means and measuring
means.
[0167] Data about the reflection light detected by the light detector 13 is send to the
analysis unit 14. From the data about the reflection light sent from the light detector
13, the analysis unit 14 extracts data about the intensity of the light reflected
from each measurement spot 6 on which a binding substance 7 is imobilized, and detects
the intensity of the reflection light for each diffraction grating surface 5a-5i of
the individual measurement spots 6. Based on the intensity of the reflection light
due to each diffraction grating surface 5a-5i, the resonance angle is calculated for
each measurement spot 6. Specifically, the diffraction grating surface whose reflection
light has the minimum intensity is selected for each measurement spot 6, and the actual
incident angle on the selected diffraction grating surface (the difference angle obtained
by subtracting the inclination angle from the incident angle on the reference plane
S0) can be treated as the resonance angle. Alternatively, it is also possible to select
plural diffraction grating surfaces adjacent to the diffraction grating surface having
the minimum reflection-light intensity, and based on both the actual incident angles
on the adjacent diffraction grating surfaces and the reflection-light intensities
of the adjacent diffraction grating surfaces, to calculate the resonance angle having
the minimum reflection-light intensity by interpolation. Of these two methods, the
method using interpolation gives more accurate calculation of the resonance angle
(step A4-1).
[0168] The analysis unit 14 compares the wavelength of the irradiation light and the calculated
resonance angle with a working curve (or a theoretical conversion equation for concentration)
to thereby analyze the concentration of the target species associated with each measurement
spot 6. The working curve is prepared preliminarily based on a test using samples
with known concentrations of target species, showing the relation between the concentration
of individual target species and each of the resonance wavelength and the resonance
angle. By comparing the calculated resonance angle at each measurement spot 6 with
the working curve, it is possible to measure the concentration of the target species
in the sample fluid (step A5-1).
[0169] By carrying out analysis using the method as described above, it becomes possible
to calculate the resonance angle at each of the plural measurement spots 6 simultaneously
in real-time, thereby real-time analysis of various target species being realized.
[0170] When the refractive index of the medium changes so largely (for example, in the case
of an enzyme reaction that produces pigmentation or other deposition on the surface,
or in the case where some minute particles such as gold colloids are used sensitizing
the binding reaction) that the shift of the resonance angle cannot be covered simply
by the variation in the angles of the diffraction grating surfaces 5a-5i, it is possible
to cope with such situation by altering the angle of the incident light.
[0171] On the other hand, in the case where the sample analysis is carried out based on
the variation of the reflection-light intensity, after the light reflected from the
sensor surface 1a is detected through the process according to the above steps A1
to A3, the analysis unit 14 extracts the data about the light reflected by each measurement
spot 6 from the obtained data about the reflection light, and detects the intensity
of the reflection light for each of the diffraction grating surfaces 5a-5i of each
measurement spot 6. Then, the analysis unit 14 determines the variation of the reflection-light
intensity from the state where when any sample is not in contact with the sensor surface
1a, for each of the diffraction grating surfaces 5a-5i (step A4-2).
[0172] Subsequently, the analysis unit 14 selects a diffraction grating surface whose variation
of the reflection-light intensity is within a predetermined determination range (determination
allowable range), in short, a diffraction grating surface that is not out of range,
for each of the measurement spots 6. Then, the analysis unit 14 compares the variation
between the reflection-light intensity of the selected diffraction grating surface,
which is not out of range, and the inclination angle of the diffraction grating surface
with a working curve (that is prepared preliminarily based on a test using samples
with known concentrations, showing the relation between the incident angle with respect
to each diffraction grating surface and the variation of the reflection-light intensity),
to thereby analyze the concentration of the target species at each spot 6 (step A5-2).
[0173] Since the wave number of the evanescent wave changes according to the incident angle
of the irradiation light with respect to the diffraction grating surface, the variation
between the reflection-light intensity also changes according to the incident angle.
Depending on the incident angle, the variation becomes so large that it exceeds the
measurement range of a measuring instrument equipped with the analysis unit 14. Conventionally,
to cope with such a situation, it is necessary to alter the incident angle through
the readjust of the optical system. Specifically in the case of carrying out the multi-item
measurement for plural target species whose concentrations differ greatly, it is required
to adjust the incident angle for each of the target species to be measured. However,
in the present embodiment, since the sensor chip 1 has plural diffraction grating
surfaces 5a-5i with different inclination angles, there is no need to actually alter
the incident angle of the irradiation light. It is possible to substantially alter
the incident angle simply by selecting another diffraction grating surface. This translates
into a substantially enlarged measurement range. As a result, it becomes possible
to deal with samples having a wide range of concentrations and therefore reqiring
a wide measurement range.
[0174] As described above, sample analysis using the sensor chip 1 of the present embodiment
has the advantages that it becomes possible to carry out real-time analysis of various
target species, and that it is also possible to deal with samples having a wide range
of concentrations.
[0175] In addition, the analysis apparatus 10 used with the sensor chip 1 is in no need
of any driving mechanism for angle scan, and its optical system can be constituted
simply of the sensor chip 1, the light source (including a polarizer) 12, and the
light detector 13. It therefore offers the advantage that simplification, miniaturization
and cost reduction of the apparatus are made possible.
[0176] In recent years, particularly in the area of clinical inspection, high importance
is placed on PoC, which is characterized by compactness and ease of operation so as
to allow on-the-spot inspection at the field of medical treatment. The surface plasmon
resonance sensor chip also has been studied for application to immunoassay and the
like, although the conventional technique is difficult to expand into PoC in respect
of size and cost. However, the present analysis apparatus 10, whose size and cost
both can be reduced, is applicable not only to PoC, but also to other areas such as
in-home inspection. Moreover, the analysis apparatus 10 is also suitable for HPLC,
and can be applied to analysis of blood or urine, analysis of nutritious substances
in food, analysis of chemical substances in drainage water, etc.
(B) Second Embodiment
[0177] Next, the second embodiment of the present invention is described with reference
to FIG. 6.
[0178] The sensor chip 21 according to the present embodiment is identical with that of
the first embodiment in its basic constitution. Namely, its surface is covered with
metal layer 23, and partly on the metal layer 23, plural diffraction areas (measurement
spots) 26 are disposed in plural positions, respectively, within each of which areas
a diffraction grating is formed.
[0179] In this embodiment, each of the measurement spots 26 has a curved surface 25 (hereinafter
called a diffraction grating surface) on which a diffraction grating is formed. When
the remaining surface on the metal layer 3 other than the measurement spots 26 is
assumed as a reference plane S0, the diffraction grating surface 25 is disposed so
as to be perpendicular to a specific plane S1 that is perpendicular to the reference
plane S0. The diffraction grating surface 25 forms a convex shape whose side facing
the sensor surface 21a bulges out, and the diffraction grating is formed thereon in
such a manner that its groove orientation is perpendicular to the specific plane S1.
[0180] With this structure, the substantial incident angle with respect to the measurement
spot 26 vaeries according to the position on the diffraction grating surface 25. In
FIG. 6, when the incident angle of the irradiation light with respect to the reference
plane S0 is assumed as θ, the actual incident angle at a position on the diffraction
grating surface 25 is represented as θ-β where β is the inclination angle that the
reference plane S0 forms with its tangent plane at the position. Since the inclination
angle of the tangent plane in contact with the diffraction grating surface 25 varies
continuously from β1 (β1>0) to β2 (β2<0) as shown in FIG. 6, the substantial incident
angle that the irradiation light forms with the diffraction grating surface 25 also
has a continuous distribution from θ-β1 to θ-β2. As a result, the reflection-light
intensity obtained at various positions on the diffraction grating surface 25 also
has a continuous distribution.
[0181] Because of these characteristics, sample analysis using the sensor chip 21 of the
present embodiment offers the same advantages as are obtained in the first embodiment,
and also offers the advantage that since the substantial incident angle of the irradiation
light with respect to the diffraction grating surface 25 has a continuous distribution,
the resonance angle can be detected directly without the need of calculations such
as approximation or interpolation, thereby analysis with higher precision being possible.
(C) Third Embodiment
[0182] Next, the third embodiment of the present invention is described with reference to
FIG. 7.
[0183] The sensor chip 31 according to the present embodiment is identical with that of
first embodiment in its basic constitution. Namely, its surface is covered with a
metal layer 33, and partly on the metal layer 33, plural diffraction areas (measurement
spots) 36 are disposed in plural positions, respectively, within each of which areas
a diffraction grating is formed.
[0184] In this embodiment, each of the measurement spots 36 has diffraction grating surfaces
35a-35i within each of which a diffraction grating is partly formed. Each of the diffraction
grating surfaces 35a-35i also has a partial area (hereinafter called non-diffraction
surface) 37a-37i within which any diffraction grating is not formed. The remaining
constitution is identical with the first embodiment.
[0185] With this structure, the reflection light from the individual measurement spot 36
includes the light that has been reflected by the diffraction grating surfaces 35a-35i
and whose intensity is therefore reduced due to a surface plasmon resonance (hereinafter
called resonance reflected light), and the light that has been reflected by the non-diffraction
surfaces 37a-37i and that is not affected by any surface plasmon resonance (hereinafter
called reference reflection light).
[0186] Because of these characteristics, when carrying out sample analysis method using
the sensor chip 31 of the present embodiment, in addition the same steps as in the
first embodiment, preferably the method further has the step of correcting the measured
intensity of the individual resonance reflection light using the intensity of the
reference reflection light from the non-diffraction surface that is on the same diffraction
grating area as the individual diffraction grating surface is on. On this account,
in addition to the same advantages as are obtained by the first embodiment, it is
possible to correct the influence of errors resulting from the difference of surface
properties between the diffraction grating surfaces by correction using the intensity
of the reference reflection light, thereby analysis with higher precision being realized.
(D) Fourth Embodiment
[0187] Next, the fourth embodiment of the present invention is described with reference
to FIG. 8.
[0188] The sensor chip 41 according to the present embodiment is identical with that of
the first embodiment in its basic constitution. Namely, its surface is covered with
a metal layer 43, and partly on the metal layer 43, plural diffraction areas (measurement
spots) 46 are disposed in plural positions, respectively, within each of which areas
a diffraction grating is formed.
[0189] In this embodiment, in addition to the binding substance 47, a non-binding substance
48 is immobilized on each of the measurement spots 46. The non-binding substance 48
is a substance that does not have the properties of binding specifically to the target
species to be detected. When plural target species are contained in the sample, it
is also preferable that both the binding substance 47 and the non-binding substance
48 are selected for each of the target species and immobilized on different measurement
spots 46.
[0190] Consequently, the area on which the binding substance is immobilized functions as
a reaction area, while the area on which the non-binding substance is immobilized
and the area on which either the binding substance or the non-binding substance is
not immobilized each function as a non-reaction area. If the metal layer 43 is composed
of a metal that does not bind specifically to the target species, it is also preferable
that any non-binding substance 48 is not immobilized, and the area on which either
the binding substance 47 or the non-binding substance 48 is not immobilized and in
which the metal layer 43 is laid bare is used as a non-reaction area.
[0191] The remaining constitution is identical with the first embodiment.
[0192] With this structure, the reflection light from the individual measurement spot 46
includes the light that has been reflected by the reaction area and the light that
has been reflected by the non-reaction area. The reflection light reflected by the
reaction area changes according to quantitative and/or qualitative factors in connection
with the sample, while the reflection light reflected by the non-reaction area is
not affected by the sample and its intensity is determined only by the structure of
the diffraction grating surface.
[0193] Because of these characteristics, when carrying out sample analysis method using
the sensor chip 41 of the present embodiment, in addition the same steps as the first
embodiment has, the method further has the step of calculating a resonance angle at
which a surface plasmon resonance occurs for each of the reaction area and the non-reaction
area, and the step of further calculating a difference resonance angle by subtracting,
from the resonance angle of the individual reaction area, the resonance angle of the
non-reaction area that is on the same diffraction grating surface as the individual
reaction area is on. On this account, since the reflection light from a reaction area
where the binding substance binds to the target species can be analyzed using the
reflection light from the non-reaction area adjacent to the reaction area as a reference,
in addition to the same advantages as are obtained by the first embodiment, it is
possible to analyze reliably the change caused by the specific binding of the binding
substance to the target species.
(E) Fifth Embodiment
[0194] Next, the fifth embodiment of the present invention is described with reference to
FIG. 9.
[0195] The present embodiment has a structure identical with that of the analysis apparatus
10 of the first embodiment, except that a separation apparatus 59 for separating a
sample fluid is disposed upstream of the flow channel 11a, through which the sample
fluid flows.
[0196] As the separation apparatus 59, it is preferable to use an apparatus that carries
out separation by a separation method such as: liquid chromatography or HPLC (high
performance liquid chromatography), in which a sample is separated according to adsorptivity
or distribution coefficient; capillary electrophoresis or microchip electrophoresis,
in which a sample is separated according to electronegativity; flow injection; or
microchannel.
[0197] The microchannel means a groove which is formed on the surface of some chip and through
which a sample flows. It is possible to carry out separation by filling a part of
the groove column with an equivalent to a filler of HPLC, or by disposing a functional
group on the surface of the groove surface.
[0198] The flow injection is a method for causing various reactions on a sample in a state
where the sample is flowing. It is possible to carry out separation by, for example,
causing complex-forming reaction, and then carrying out solvent extraction and an
additional process such as the removal of substances other than the target species
in the sample.
[0199] It is also possible to install an apparatus other than above in the analysis apparatus
as the separation apparatus.
[0200] When carrying out analysis using the apparatus, it is possible to separate a sample,
which contains a target species such as an enzyme or a protein, into elements with
higher purity in advance by the separation apparatus. Consequently, it is possible
to analyze the target species in a purer form, thereby analysis with higher precision
being made possible.
[0201] Further, by adopting a generally used detection method (absorbance detection, fluorescence
detection, chemiluminescence detection, differential refract meter detection, electrochemical
detection, etc.) as the analyzing means, it becomes possible to carry out both the
measurement of the amounts of various substances existing in the sample and the measurement
of object species to be detected among these substances by specific reaction at a
time.
(F) Sixth Embodiment
[0202] Next, the sixth embodiment of the present invention is described with reference to
FIG. 5.
[0203] The basic constitution of the present embodiment is identical with that of the first
embodiment. Namely, it is constituted in such a manner that the light emmited from
the light source 12 is reflected by the sensor chip and detected by the light detecting
unit 13.
[0204] In this embodiment, the target species is a light-emitting substance, which can produce
light such as fluorescence or phosphorescence. Examples of the target species include
a light-emitting substance that emits light by reaction with the binding substance,
and a light-emitting substance that emits light under excitation by the light supplied
from the light source 12. The present embodiment is constituted in such a manner that
the light detecting unit 13 can detect the light emitted (emission light).
[0205] With this constitution, in addition to the same advantages as are obtained in the
first embodiment, the present embodiment offers the additional advantage that it is
possible to quantitatively and/or qualitatively analyze the sample using the detection
result of the emission light, analysis with higher precision therefore being realized.
Chemiluminescence such as fluorescence, in particular, is highly sensitive and is
therefore especially useful to detect minute reaction.
(G) Seventh Embodiment
[0206] Next, the seventh embodiment of the present invention is described with reference
to FIGs. 10 and 11.
[0207] The basic constitution of the present embodiment is identical with that of the first
embodiment. Namely, it is constituted in such a manner that the light emitted from
the light source 12 is reflected by the sensor chip 71 and detected by the light detecting
unit 13.
[0208] As shown in FIG. 10, this embodiment has plural flow channels 70, through each of
which a sample flows, laid in a direction orthogonal to an uneven surface being a
diffraction grating. The flow channels 70 are disposed between a cover 72 covering
the surface of the sensor chip 71 and the sensor chip 71, and are formed in such a
manner that they pass in pairs on each measurement spot 6 on the sensor chip 71. Interstices
between each two adjacent flow channels 70 are filled with seals such that the samples
do not become mixed.
[0209] Moreover, as shown in FIG. 11, the present embodiment also has a sample introducing
apparatus 79, disposed upstream of the flow channels 70 of the analysis apparatus
10, for assigning different sample fluids to the flow channels 70, respectively, and
for introducing each of the assined sample fluids to the respective flow channel.
[0210] With this constitution, in addition to the same steps as the first embodiment has,
the method according to the present embodiment further has the step of assigning plural
different samples to the plural flow channels 70, respectively, and irradiating the
sensor surface with light while making each of the assigned samples flow through the
respective flow channel 70.
[0211] On this account, in addition to the same advantages as are obtained in the first
embodiment, the present embodiment also offers the advantage that since plural samples
can be analyzed at a time, it is possible to carry out analysis work with efficiency.
It further offers the additional advantage that since the plural sample can be alanyzed
under the same conditions, it is possible to analyze differences between the samples
clearly.
(H) Eighth Embodiment
[0212] Next, the eighth embodiment of the present invention is described with reference
to FIG. 12.
[0213] The sensor chip according to the present embodiment 81 is identical with that of
the first embodiment in its basic constitution. Namely, its surface is covered by
the metal layer 83, and partly on the metal layer 83, plural diffraction areas (measurement
spots) 87 are disposed in plural positions, respectively, within each of which areas
a diffraction grating is formed.
[0214] In this embodiment, a non-diffraction area 88 is disposed adjacent each of the measurement
spots 87. The non-diffraction area 88 has plural planes (hereinafter called non-diffraction
surfaces) on each of which any diffraction grating is not formed. The inclination
angles that the non-diffraction surfaces form with the reference plane S0 have the
same distribution as that of the inclination angles of the diffraction grating surfaces
of the measurement spot 87 with respect to the reference plane S0. The remaining constitution
is identical with that of the first embodiment.
[0215] With this structure, the reflection light from the sensor chip 81 includes a resonance
reflection light, which has been reflected by the measurement spot 87 and whose intensity
is therefore reduced by a surface plasmon resonance, and a reference reflection light,
which has been reflected by the non-diffraction area 88 and which is therefore not
affected by any surface plasmon resonance.
[0216] Because of these characteristics, when carrying out sample analysis method using
the sensor chip 81 of the present embodiment, in addition to the same steps as the
first embodiment has, the method further has the step of correcting the measured intensity
of the resonance reflection light from the individual measurement spot, using the
intensity of the reference reflection light reflected by the non-diffraction area
corresponding to the individual measurement spot. On this account, in addition to
the same advantages as are obtained by the first embodiment, the present embodiment
offers the advantage that it is possible to correct the influence of errors resulting
from the difference of surface properties between the diffraction grating surfaces
formed on the surface of each measurement spot by correction using the intensity of
the reference reflection light, thereby analysis with higher precision being realized.
(I) Ninth Embodiment
[0217] Next, the ninth embodiment of the present invention is described with reference to
FIG. 13.
[0218] The sensor chip according to the present embodiment 91 is identical with that of
the first embodiment in its basic constitution. Namely, its surface is covered with
a metal layer 93, and partly on the metal layer 93, plural diffraction areas (measurement
spots) 96 are disposed in plural positions, respectively, within each of which areas
a diffraction grating is formed.
[0219] In this embodiment, a binding substance 97 is immobilized on the surfaces of some
measurement spots 96, while a non-binding substance 98 is immobilized on the surfaces
of the remaining measurement spots 96. The non-binding substance 98 is a substance
that does not have the properties of binding specifically to the target species to
be detected. When plural target species are contained in the sample, it is also preferable
that both the binding substance 97 and the non-binding substance 98 are selected for
each of the target species and immobilized on different measurement spots 96.
[0220] Consequently, in each of the measurement spots having binding-substance immobilized
surfaces, the area on which the binding substance is immobilized serves as a reaction
area, while the area on which the non-binding substance is immobilized and the area
on which either any binding substance or any non-binding substance is not immobilized
each serves as a non-reaction area.
[0221] Also, if the metal layer 93 is composed of a metal that does not bind specifically
to the target species, it is also preferable that any non-binding substance 98 is
not immobilized, and the area on which either the binding substance 97 and the non-binding
substance 98 is not immobilized and in which the metal layer 93 is laid bare is used
as a non-reaction area.
[0222] The remaining constitution is identical with the first embodiment.
[0223] With this structure, the reflection light from the measurement spots 96 includes
the light that has been reflected by the reaction areas and the light that has been
reflected by the non-reaction areas. The reflection light reflected from the reaction
areas changes according to quantitative and/or qualitative factors connected with
the sample, while the reflection light reflected by the non-reaction areas is not
affected by the sample and its intensity is determined only by the structure of the
diffraction grating surface.
[0224] Because of these characteristics, when carrying out sample analysis method using
the sensor chip 91 of the present embodiment, in addition to the same steps as the
first embodiment has, the method further has the step of calculating a resonance angle
at which a surface plasmon resonance occurs for each of the reaction area and the
non-reaction area, and the step of further calculating a difference resonance angle
by subtracting, from the resonance angle of the individual reaction area, the resonance
angle of the non-reaction area that is on the same diffraction grating surface as
the individual reaction area is on. On this account, since the light reflected from
a reaction area in which the binding substance binds to the target species can be
analyzed using the light reflected from a non-reaction area which is adjacent to the
reaction area as a reference, in addition to the same advantages as are obtained by
the first embodiment, it is possible to analyze reliably the change caused by the
specific binding of the binding substance to the target species.
(J) Tenth Embodiment
[0225] Next, the tenth embodiment of the present invention is described with reference to
FIGs. 11 and 14.
[0226] The basic constitution of the present embodiment is identical with that of the first
embodiment. Namely, it is constituted in such a manner that the light emitted from
the light source 12 is reflected by the sensor chip 101 and detected by the light
detecting unit 13.
[0227] As shown in FIG. 14, this embodiment has plural flow channels 100, through each of
which a sample flows, disposed side by side on the surface of the sensor chip 101.
The flow channels 100 are disposed between a cover 102 covering the surface of the
sensor chip 101 and the sensor chip 101. Interstices between each two adjacent flow
channels 100 are filled with seals such that the samples do not become mixed.
[0228] This embodiment further has plural measurement spots 6 so as to be associated with
each of the flow channels 100, which spots are the same as those of the first embodiment.
[0229] Moreover, as shown in FIG. 11, the present embodiment also has a sample introducing
apparatus 79, disposed upstream of the flow channels 100 of the analysis apparatus
10, for assigning different sample fluids to the flow channels 100, respectively,
and for introducing each of the assined sample fluids to the respective flow channel.
[0230] With this constitution, in addition the same steps as the first embodiment has, the
method according to the present embodiment also has the step of assigning plural different
samples to the plural flow channels 100, respectively, and irradiating the sensor
surface with light while making each of the assigned samples flow through the respective
flow channel 100.
[0231] On this account, in addition to the same advantages as are obtained in the first
embodiment, the present embodiment also offers the advantage that since plural samples
can be analyzed at a time, it is possible to carry out the work with efficiency. It
further offers the additional advantage that since the plural sample can be alanyzed
under the same conditions, it is possible to analyze differences between the samples
clearly.
[0232] It is also preferable to carry out the present embodiment in combination with the
fourth embodiment or the ninth embodiment. On this account, since both a reaction
area and a non-reaction area can be formed for each of the diffraction grating surfaces
on the same flow channel, it is possible to carry out correction for each of the samples
flowing through the respective flow channel, using the reflection light from each
of the non-reaction areas.
(K) Eleventh Embodiment
[0233] At first, the constitution of a sensor chip (surface plasmon resonance sensor chip)
201 according to the eleventh embodiment of the present invention is described with
reference to FIG. 15. As shown in FIG. 15, the sensor chip 201 according to the present
embodiment is constituted such that its surface (sensor surface) 201a is covered with
a metal layer 203, and that a diffraction grating 205 is formed on the metal layer
203. In the present embodiment, the diffraction grating 205 is constituted in such
a manner that its groove pitches are not uniform but have a discontinuous distribution.
Specifically, the diffraction grating 5 has four areas 251, 252, 253, 254 disposed
continuously in a direction perpendicular to the grooves. The areas 251, 252, 253,
254 have different groove pitches in such a manner that their groove pitches are positioned
in ascending order from the area 251 to the area 254 (namely, d1 < d2 < d3 < d4).
Each of these areas 251, 252, 253, 254 is used as a diffraction grating surface according
to the present invention.
[0234] With this structure, the sensor surface 201a of the sensor chip 201 is irradiated
with light, the irradiation light is diffracted by the diffraction grating 205 on
the sensor surface 201a, and the diffraction phenomenon generates an evanescent wave.
The irradiation light also affects the metal layer 203 to generate a surface plasmon
wave along the surface of the metal layer 203. When the irradiation light having a
specific wavelength and a specific incident angle, the evanescent wave resonates with
the surface plasmon wave to produce surface plasmon resonance (SPR). That is to say,
in the sensor chip 201, the area (continuous area) on the metal layer 203 in which
the diffraction grating 205 is formed serves as a resonance area.
[0235] When using the sensor chip 201 (for sample analysis), as shown in FIG. 16, a binding
substance 206, 207 is immobilized on the resonance area, in which the diffraction
grating 205 is formed, so that the resonance area serves as the reaction area for
reacting with the target species (chemical species, biochemical species, biological
species, etc.) in the sample. The binding substance 206, 207 is a substance that has
the property of binding specifically to a specific substance by some interaction such
as antigen-antibody reaction, complementarily binding of DNA, receptor-ligand interaction,
or enzyme-substrate interaction. The binding substance 206, 207 is selected in accordance
with a target species to be detected. With this embodiment, two binding substances
206, 207 associated with different target species are immobilized on each of the areas
251-254.
[0236] The diffraction grating formed on the sensor chip has a groove pitch between 200nm
and 2000nm, preferably between 500nm and 900nm, and a groove depth preferably between
10nm and 100nm. The number of varieties of groove pitches formed on the single sensor
chip is between 1-200, preferably between 2-20.
[0237] Meanwhile, the surface plasmon wave induced along the metal layer 203 upon light
irradiation is represented by the following formula 1,
where the wave number is k
sp [k
sp = 2π/λ
sp (wavelength)].
[0238] Also, the evanescent wave generated by the action of the diffraction grating 205
is represented by the following formula 2,
where wave number is k
ev [k
ev = 2π/λ
ev (wavelength)].
[0239] In the formula 1 and formula 2, ω means the angular frequency of the irradiation
light, θ means the incident angle of the irradiation light, k
g means the grating constant of the diffraction grating 205, m means the diffraction
order of the diffraction light by the diffraction grating 205, ε
m(ω) means the dielectric constant of the metal layer 203, n
1(ω) means the refractive index of the medium adjacent to the metal layer 203 surface,
and c means the light speed.
[0240] Plotting the relations defined by the above formulae 1 and 2 on the same graph gives
the dispersion relationship diagram shown in FIG. 17.
[0241] In FIG. 17, the curved lines A1, A2 each show the relation between the angular frequency
ω and the wave number k of the surface plasmon wave. As is evident from formula 1,
the surface plasmon wave denpends on the refractive index of the medium adjacent to
the metal layer 203 surface: when the refractive index of the medium becomes larger,
the curved line showing the relation between the angular frequency ω and the wave
number k of the surface plasmon wave also changes. Specifically, as shown in FIG.
16, when each of the binding substances 206, 207 is immobilized in a spot on the metal
layer 203 surface and binds to the associated target species, the refractive index
of the individual spot varies according to the refractive index and the amount of
the target species that is bound to the spot. In this figure, the spot on which the
binding substance 206 is immobilized has a higher refractive index than that of the
spot on which the binding substance 207 is immobilized. Namely, the curved line A1
corresponds to the spot on which the binding substance 206 is immobilized while the
curved line A2 corresponds to the spot on which the binding substance 207 is immobilized.
[0242] In the meantime, the straight line B0 in FIG. 17 shows the relation between the angular
frequency ω and the wave number k of the irradiation light, while the straight lines
B1-B4 each show the relation between the angular frequency ω and the wave number k
of the evanescent wave. As is evident from formula 2, the relation between the angular
frequency ω and the wave number k of the evanescent wave varies according to the grating
constant k
g, which grating constant k
g is determined by the substantial groove pitch of the diffraction grating 205 when
viewed from the direction of incidence of light. The narrower the substantial groove
pitch, the larger the grating constant k
g. In the figure, the straight line B1 corresponds to the evanescent wave generated
in the area 251 having the narrowest groove pitch, the straight line B2 corresponds
to the area 252, the straight line B3 corresponds to the area 253, and the straight
line B4 corresponds to the area 254. Each of the intersection points P11, P12, P21,
P22, P31, P32, P41, P42 between the curved lines A1, A2, mentioned above, and the
straight lines B1-B4 indicates a resonance point at which surface plasmon resonance
occurs.
[0243] It is possible to analyze the concentration of the target species in the sample by
detecting a resonance phenomenon at the spot in which the associated binding substance
206, 207 is immobilized. However, when the measurement range (measurement wavelength
area) of a measuring instrument is limited, not every resonance phenomenon can be
detected. Suppose the measurement range of angular frequency is limited, for example,
to the range from ω1 to ω2. Regarding the areas 251-253, in which the groove pitch
of the diffraction grating 205 is narrow, since the resonance points P12, P22, P32,
fall outside the measurement range, it is impossible to detect a resonance phenomenon
at the spot in which the binding substance 207 is immobilized. On the other hand,
regarding the area 254, which has the broader groove pitch d4, since the resonance
point P42 falls within the measurement range, it is possible to detect a resonance
phenomenon at the spot in which the binding substance 207 is immobilized. Likewise,
when a resonance phenomenon occurs at the spot in which the binding substance 206
is immobilized, it is impossible to detect the resonance phenomenon in the areas 253,
254 since the resonance points P31, P41 fall outside the measurement range, while
it is possible to detect the resonance phenomenon in the areas 251, 252 since the
resonance points P11, P21 fall within the measurement range.
[0244] Consequently, since the sensor chip 201 has the diffraction grating 205 whose groove
pitch differs depending on the areas 251-254, the resonance points at which a resonance
phenomenon occurs in the areas 251-254 are distributed over a dispersion curve of
the surface plasmon wave. It is therefore possible to detect a resonance phenomenon
at any of the areas 251-254 with a high probability even if the measurement range
is limited. Further, even if plural kinds of binding substances 206, 207 are immobilized
to analyze plural different target species having different refractive indexes, as
mentioned above, it is possible to carry out analysis at a time within a limited measurement
range without the need of readjusting the optical system. In short, it is possible
to analyze a sample whose refractive indexes have a wide distribution.
[0245] It is possible to make the sensor chip 201 by the following processes. Using a substrate
202 shown in FIG. 18(a), the first process is, as shown in FIG. 18(b), to make an
uneven form (grating) 204 by laser processing or the like. The second process is,
as shown in FIG. 18(c), to laminate all over the surface of the substrate 202 with
a metal layer 203 by sputtering, vapor deposition, or the like. Since the metal layer
203 is layered on the uneven form 204, the surface of the metal layer 203 also has
an uneven form thereon. This uneven form on the metal layer 203 surface functions
as the diffraction grating 205.
[0246] The material used for the substrate 202 is not limited as long as it allows the uneven
form 204 to be made on its surface and as it possesses adequate mechanical strength
to hold the metal layer 203. Examples of usable inorganic materials include glass,
quartz, and silicon, while examples of usable organic materials include various resin
such as poly(methyl methacrylate), polycarbonate, polystyrene, or polyolefin. When
making the uneven form 204 on the substrate 202, it is required to consider the thickness
of the metal layer 203 or the likes in such a manner that when the metal layer 203
is layered, a desired diffraction grating 205 is obtained on the surface. As a method
of making the uneven form, in addition to the laser processing mentioned above, it
is also use other methods such as injection molding, compression molding, or etching.
The uneven form 204 can be a square-wave form, a sine-wave shape, a saw-tooth shape,
etc., although being not limited as long as it can induce a diffraction phenomenon
along the diffraction grating 205 to generate an evanescent wave. It is required to
vary the groove pitches of the uneven form 204 among the four continuous areas 241,
242, 243, 244: the groove pitch in the area 241 is made d1, the groove pitch in the
area 242 is made d2, the groove pitch in the area 243 is made d3, and the groove pitch
in the area 244 is made d4. Meanwhile, the depth of the uneven form 204 (depth of
the diffraction grating 205) is related with the peak's sharpness of the reflection-light
intensity and therefore has an influence on resolution.
[0247] The material used for the metal layer 203 is not limited as long as it can induce
a surface plasmon wave. Examples of the usable material include gold, silver, and
aluminum. Among above, silver is preferable in respect of sensitivity and inexpensiveness,
while gold is preferable in point of stability. If the thickness of the metal layer
203 is too small, the irradiation light passes through the metal layer 203 and reaches
the surface of the substrate 202, and may be diffracted by the uneven form 204 on
the substrate 202 surface. In this case, the uneven form 204 functions as a diffraction
grating.
[0248] A method of applying the metal layer 203 on the substrate 202 is not limited as long
as it can bind the substrate 202 with the metal layer 203 tightly to a sufficient
degree. Representative examples include vapor deposition, sputtering, and plating.
It is also preferable to use some other substance between the substrate 202 and the
metal layer 203 in order to, for example, improve adhessiveness. Moreover, it is also
preferable to coat the surface of the metal layer 203 with a layer such as coating
film or an activated film to thereby make the surface of the metal layer 203 serve
some function.
[0249] Meanwhile, the sensor chip 1 can be embodied with some modification in such a form
as the sensor chip 201' shown in FIG. 19. Namely, instead of disposed continuously,
the areas 251-254 having different groove pitches of the diffraction grating 205 are
disposed discretely on the metal layer 23 in such a manner that they have the same
groove orientation. With this structure, it is also possible to achieve the same operations
and advantages as achieved by the sensor chip 201.
[0250] Then, a method for using the sensor chip 201 according to the present embodiment
is described.
[0251] When using the sensor chip 1 for sample analysis, as shown in FIG. 16, binding substances
206, 207 are immobilized on the sensor surface 201a in advance. Then the sensor chip
201, on which the binding substances 206, 207 are immobilized, is set on an analysis
apparatus 210 having the constitution as shown in FIG. 20 while analysis is carried
out. The analysis apparatus 210 is constituted basically of a holder (holding means)
211 for holding the sensor chip 201, a light source (light irradiation means) 212,
a light detector (light receiving means) 213, and an analysis unit 214.
[0252] The holder 211 has a flow channel 211a formed thereon, through which a sample fluid
containing a target species passes. The sensor chip 201 is disposed and fixed in such
a manner that the sensor surface 201a is in contact with the sample flowing through
the flow channel 211a.
[0253] The light source 212 is disposed opposite to the sensor chip 201 across the flow
channel 211a so as to irradiate the sensor surface 201a of the sensor chip 201 with
light. As the light source 212, it is possible to use either a monochromatic light
source or a multicomponent light source (such as a white light source). Also the light
can be either coherent or not. As the monochromatic light source, it is desirable
to use a laser light source, and specifically, a semiconductor laser in respect of
cost and size. It is also possible to spectrally separate white light, which is emitted
from a halogen tungsten lamp or others, using interference filter, spectroscope, or
the like to thereby obtain monochromatic light for the light source purpose. As the
white light source, it is preferable to use a halogen tungsten lamp, a xenon lamp,
etc.
[0254] The light detector 213 is a detector for detecting the reflection light from the
sensor chip 201. As the light detector 213, it is desirable to use, for example, integrated
CCD elements, a silicon photodiode array, etc. In addition, although not illustrated
in the figure, a polarizer is disposed between the light source 212 and the sensor
chip 201 or between the sensor chip 201 and the light detector 213, so as to polarize
the irradiation light from the light source 212 or the reflection light from sensor
chip 201, since only polarized light P can cause resonance with a surface plasmon
wave.
[0255] The analysis unit 214 is an apparatus for carrying out analysis processing based
on the detection data from the light detector 213. The analysis unit 214 functions
as measuring means and also as analyzing means according to the present invention.
The following description is given to the detailed explanation of the individual functions
of analysis unit 214, and also to a sample analysis procedure using the sensor chip
201 according to the present embodiment.
[0256] The first process is to set the sensor chip 201 to the holder 211 and to make the
sensor surface 201a of the sensor chip 201 in contact with the sample (step B1). Thereby
the binding substance 206, 207 imobilized on the sensor surface 201a comes to bind
specifically to the target species contained in the sample fluid According to the
amount of substance of the target species, the refractive index of a medium adjacent
to the surface of metal layer 203 changes on each of the spots in which the binding
substance 206, 207 is immobilized, and conditions under which the resonance of a surface
plasmon wave occurs at each spot also change accordingly.
[0257] The second process is to irradiate the sensor surface 201a with monochromatic light
from the light source 212 at a constant irradiation angle (step B2). At the time,
the area of the irradiation light is adjusted such that the irradiation light illuminates
all the spots on which the binding substance 206, 207 is immobilized. With this feature,
it is possible to measure all the spot simultaneously. The irradiation light entered
to the sensor surface 201a produces diffraction lights on the diffraction grating
205. Among these diffraction lights, the light detector 213 detects the diffraction
light of the 0th-order (reflection light) (step B3).
[0258] Data about the reflection light detected by the light detector 213 is send to the
analysis unit 214. From the data about the reflection light sent from the light detector
213, the analysis unit 214 extracts data about the intensity of the light reflected
from each of the spots on which the binding substance 206, 207 is immobilized, and
measures the intensity of the reflection light for each spot. Based on the intensity
of the reflection light due to each spot, the groove pitch of the diffraction grating
205 with which a resonance phenomenon occurs is detected for each of the target species
(for the individual binding substances 206, 207). Specifically, the spot having the
minimum intensity of the reflection light is detected for each target species, and
it is assumed that a resonance phenomenon occurs at the detected spot. Alternatively,
based on the intensity of the reflection light at each spot, it is also possible to
calculate the groove pitch with which a resonance phenomenon occurs by interpolation
using software (step B4-1).
[0259] The analysis unit 214 compares the detected groove pitch, with which a resonance
phenomenon occurs, with a working curve (or a theoretical conversion equation for
concentration), which is prepared preliminarily based on measurement, to thereby analyze
the concentration of the individual target species in the sample. In this embodiment,
the analysis unit 214 functions as the analyzing means according to the present invention
(step B5-1).
[0260] Meanwhile, it is also possible to carry out sample analysis based on the variation
of the reflection-light intensity. The distribution of the reflection-light intensity
over the incident angle or the incident wavelength changes according to the amount
of the target species captured by the binding substance, namely, the concentration
of the target species in the sample. On this account, the method is characterized
by measuring the intensity of reflection light under conditions of a constant incident
angle and a constant incident wavelength, and analyzing the concentration and other
properties based on the variation of the intensity under the conditions (from the
state of not being in contact with any sample). In this case, after the light reflected
from the sensor surface 201a is detected according to the above steps B1 to B3, the
analysis unit 214 extracts the data about the light reflected by each spot on which
the binding substance 206, 207 is immobilized from the data about the reflection light,
and detects the intensity of the reflection light for each of the areas 251-254. Then,
the analysis unit 214 determines the variation of the reflection-light intensity from
the state where when any sample is not in contact with the sensor surface 201a, for
each area 251-254 (step B4-2).
[0261] Subsequently, the analysis unit 214 selects an area (diffraction grating) whose variation
of the reflection-light intensity is within a predetermined determination range (determination
allowable range), in short, an area that is not out of range. Then, the analysis unit
214 compares the variation of the reflection-light intensity of the selected area,
which is not out of range, and the groove pitch of the area with a working curve (that
is prepared preliminarily based on a test using samples with known concentrations,
showing the relation between the groove pitch and the variation of the reflection-light
intensity), to thereby analyze the concentration of the target species in the sample
(step B5-2).
[0262] Since the wave number of the evanescent wave changes according to the incident angle
of the irradiation light with respect to the diffraction grating surface, the variation
of the reflection-light intensity also changes according to the incident angle. Depending
on the incident angle, the variation becomes so large that it exceeds the measurement
range of a measuring instrument equipped with the analysis unit 214. Conventionally,
to cope with such a situation, it is necessary to alter the incident angle through
the readjust of the optical system. Specifically in the case of carrying out the multi-item
measurement for plural target species whose concentrations differ greatly, it is required
to adjust the incident angle for each of the target species to be measured. However,
in the present embodiment, since the sensor chip 201 has plural areas (diffraction
grating surface) 251-254 with different groove pitches, there is no need to actually
alter the incident angle of the irradiation light. It is possible to substantially
alter the incident angle simply by selecting another area (diffraction grating surface).
This translates into a substantially enlarged measurement range. As a result, it becomes
possible to deal with samples having a wide range of concentrations and therefore
reqiring a wide measurement range.
[0263] Analysis according to the above method eliminates the need for complicated operations
such as the readjustment of optical system, which are necessary for the conventional
method. Further, it becomes possible to carry out sample analysis with simple apparatus
constitution compared to the conventional angle-change type, specifically, with simple
optical system. Meanwhile, the above steps B1-A5 can be carried out not only in sequence,
as described above, but also simultaneously. When the steps are carried out simultaneously,
it becomes possible to monitor the manner in which the target species in the sample
binds to the binding substance 206, 207 in real time.
[0264] Whereas the above description has been made of a method in which the sensor chip
is irradiated with light of a single wavelength at a single angle (fixed angle), it
is also possible to apply the sensor chip 201 of the present embodiment to a method
in which the sensor chip is irradiated with light of a single wavelength while the
irradiation angle is varied. In the method, the first process is to measure (or interpolatively
calculate) a groove pitch with which a resonance phenomenon occurs, based on the reflection-light
intensity. Then, the second process is to analyze the concentration of the target
species in the sample using a working curve (or a theoretical conversion equation
for concentration) which is preliminaliry obtained through measurement, based on both
the measured groove pitch with which a resonance phenomenon occurs and the intensity
of the reflection light in a position where a resonance phenomenon is detected (in
fact, the resonance angle determined from the reflection-light intensity).
[0265] Meanwhile, a monochromatic light source is used as the light source 212 in the case
described above. On the other hand, using a white light source as the light source
212, it is possible to detect all the resonance points P11, P21, P42 existing within
a measurement range (ω1-ω2) at a time, as shown in FIG. 17. Specifically, the concentration
of a target species associated with the binding substance 206 can be analyzed by using
a working curve (or a theoretical conversion equation for concentration) which is
preliminaliry obtained through measurement, based on both the groove pitch d1 corresponding
to the resonance point P11 and the intensity of the reflection light in a position
(area 251) where a resonance phenomenon is detected (in fact, the resonance wavelength
determined from the reflection-light intensity), or altenatively, based on both the
groove pitch d2 corresponding to the resonance point P21 and the intensity of the
reflection light in a position (area 252) where a resonance phenomenon is detected.
Contrarily, the concentration of a target species associated with the binding substance
207 can be analized based on both the groove pitch d4 corresponding to the resonance
point P42 and the intensity of the reflection light in a position where a resonance
phenomenon is detected (area 254).
(L) Twelfth Embodiment
[0266] Next, the twelfth embodiment of the present invention is described with reference
to FIG. 21(a). In this figure, substantially identical components with the eleventh
embodiment are indicated by the same reference numerals.
[0267] Like the eleventh embodiment, the sensor chip 261 according to the present embodiment
is identical with that of the conventional sensor chip in its basic constitution.
Namely, its surface (sensor surface) 261a is covered with a metal layer 203, and a
diffraction grating 205 is formed on the metal layer 203.
[0268] In this embodiment, each of the areas 251, 252, 253, 254 with different groove pitches
also has a partial area (hereinafter called a non-diffraction surface) 251x, 252x,
253x, 254x within which the diffraction grating 205 is not formed. The remaining constitution
is identical with the eleventh embodiment.
[0269] With this structure, the reflection light from the sensor chip 261 includes the light
that has been reflected by the areas 251-254, in which the light diffraction grating
205 is formed, and whose intensity is therefore reduced due to a surface plasmon resonance
(hereinafter called resonance reflection light) and the light that has been reflected
by the non-diffraction surfaces 251x-254x and that is therefore not affected by any
surface plasmon resonance (hereinafter called reference reflection light).
[0270] Because of these characteristics, when carrying out sample analysis method using
the sensor chip 261 of the present embodiment, in addition the same steps as in the
eleventh embodiment, preferably the method further has the step of correcting the
measured intensity of the individual resonance reflection light using the resonance
reflection light intensity of the reference reflection light from the non-diffraction
surface that is on the same diffraction grating area as the individual diffraction
grating surface is on. On this account, in addition to the same advantages as are
obtained by the eleventh embodiment, the embodiment offers the additional advantage
that it is possible to correct the influence of errors resulting from the difference
of surface properties between the diffraction grating surfaces by correction using
the intensity of the reference reflection light, thereby analysis with higher precision
being realized.
[0271] The present embodiment is further explained with reference to FIG. 21(b). FIG. 21(b)
is a schematic diagram showing the relation between surface structure of the sensor
chip 261 and reflection-light intensity.
[0272] The sensor chip 261 has the areas 251-254 on its surface, as described above, and
the diffraction grating is formed over these areas so as to have different groove
pitches between these areas. The areas 251-54 also have different surface properties.
Most of the differences of surface properties are minute differences due to manufacturing
stage of the sensor chip, but they still have an influence on the intensity of the
light reflected from the sensor chip despite their minuteness. Accordingly, the reflection
lights from the areas 251-254 also differ.
[0273] The sensor chip 261 of the present invention is intended for the analysis of a sample
placed on the surface of the sensor chip 261, based on a resonance between a surface
plasmon wave and an evanescent wave. It is therefore undesirable that variation in
reflection light arises due to differences in surface properties of the sensor chip
261 independently of properties of the sample.
[0274] For that reason, in the present embodiment, the intensity of the reflection light
from each of the areas 251-254 is corrected using the intensity of the reflection
light from the associated area 251x-254x, which is disposed in each area 251-254 and
does not have any diffraction grating.
[0275] In FIG. 21(b), for example, difference between the intensity of the reflection light
from the area 251 and the intensity of the reflection light from the area 251x is
assumed as the the reflection-light intensity reduced by a resonance between a surface
plasmon wave and an evanescent wave.
[0276] By carrying out such a correction as discussed above, analysis with higher precision
is made possible.
(M) Thirteenth Embodiment
[0277] Next, the thirteenth embodiment of the present invention is described with reference
to FIG. 22. In this figure, substantially identical components with the first and
second embodiments are indicated by the same reference numerals.
[0278] Like the eleventh embodiment, the sensor chip according to the present embodiment
271 is identical with that of the conventional sensor chip in its basic constitution.
Namely, its surface (sensor surface) 271a is covered with a metal layer 203, and a
diffraction grating 205 is formed over plural areas 251-254 disposed on the metal
layer 203.
[0279] In this embodiment, in addition to the binding substance 206, a non-binding substance
291 is immobilized on the areas 251-254. The non-binding substance 291 is a substance
that does not have the properties of binding specifically to the target species to
be detected, and is selected in accordance with the target species to be detected
(chemical species, biochemical species, biological species, etc.). When the sample
contains plural target species, plural binding substances 206 and plural non-binding
substances 291 are selected according to the plural target species, respectively,
and immobilized on different areas 251-254. Consequently, the area on which the binding
substance 206 functions as a reaction area, while the area on which the non-binding
substance 291 is immobilized and the area on which either the binding substance 206
or the non-binding substance 291 is not immobilized each function as a non-reaction
area. If the metal layer 203 is composed of a metal that does not bind specifically
to the target species, it is also preferable that any non-binding substance 2091 is
not immobilized, and the area on which either the binding substance 206 or the non-binding
substance 291 is not immobilized and in which the metal layer 203 is laid bare is
used as a non-reaction area.
[0280] The remaining constitution is identical with that of the first embodiment.
[0281] With this structure, the reflection light from the individual area 251-254 includes
the light that has been reflected by the reaction area and the light that has been
reflected by the non-reaction area. The reflection light reflected by the reaction
area changes according to quantitative and/or qualitative factors in connection with
the sample, while the reflection light reflected by the non-reaction area is not affected
by the sample and its intensity is determined only by the structure of the diffraction
grating surface.
[0282] Because of these characteristics, when carrying out sample analysis method using
the sensor chip 271 of the present embodiment, in addition the same steps as in the
eleventh embodiment, the method further has the step of identifying a groove pitch
with which a surface plasmon resonance occurs for each of the reaction area and the
non-reaction area, and the step of analyzing based on the groove pitch of the reaction
area and the non-reaction area. On this account, since the reflection light from a
reaction area where the binding substance binds to the target species can be analyzed
using the reflection light from the non-reaction area adjacent to the reaction area
as a reference, in addition to the same advantages as are obtained by the eleventh
embodiment, it is possible to analyze reliably the change caused by the specific binding
of the binding substance to the target species.
(N) Fourteenth Embodiment
[0283] Next, the fourteenth embodiment of the present invention is described with reference
to FIG. 23. In this figure, substantially identical components with the eleventh through
thirteenth embodiments are indicated by the same reference numerals.
[0284] The present embodiment has a structure identical with that of the analysis apparatus
210 of the eleventh embodiment, except that a separation apparatus 292 for separating
a sample fluid is disposed upstream of the flow channel 211a, through which the sample
fluid flows.
[0285] As the separation apparatus 292, it is preferable to use an apparatus that carries
out separation by a separation method such as: liquid chromatography or HPLC (high
performance liquid chromatography), in which a sample is separated according to adsorptivity
or distribution coefficient; capillary electrophoresis or microchip electrophoresis,
in which a sample is separated according to electronegativity; microchannel; or flow
injection. It is also possible to install an apparatus other than above in the analysis
apparatus as the separation apparatus.
[0286] The microchannel means a groove which is formed on the surface of some chip and through
which a sample flows. It is possible to carry out separation by filling a part of
the groove column with an equivalent to a filler of HPLC, or by disposing a functional
group on the surface of the groove surface.
[0287] The flow injection is a method for causing various reactions on a sample in a state
where the sample is flowing. It is possible to carry out separation by, for example,
causing complex-forming reaction, and then carrying out solvent extraction and an
additional process such as the removal of substances other than the target species
in the sample.
[0288] It is also possible to install an apparatus other than above in the analysis apparatus
as the separation apparatus.
[0289] When carrying out analysis using the apparatus, it is possible to separate a sample,
which contains a target species such as an enzyme or a protein, into elements with
higher purity in advance by the separation apparatus. Consequently, it is possible
to analyze the target species in a purer form, thereby analysis with higher precision
being made possible.
[0290] Further, by adopting a generally used detection method (absorbance detection, fluorescence
detection, chemiluminescence detection, differential refract meter detection, electrochemical
detection, etc.) as the analyzing means, it becomes possible to carry out both the
measurement of the amounts of various substances existing in the sample and the measurement
of object species to be detected among these substances using specific reaction at
a time.
(O) Fifteenth Embodiment
[0291] Next, the fifteenth embodiment of the present invention is described with reference
to FIG. 20. In this figure, substantially identical components with the eleventh through
fourteenth embodiments are indicated by the same reference numerals.
[0292] The basic constitution of the present embodiment is identical with that of the eleventh
embodiment. Namely, it is constituted in such a manner that the light emmited from
the light source 212 is reflected by the sensor chip and detected by the light detecting
unit 213.
[0293] In this embodiment, the target species is a light-emitting substance, which can produce
light such as fluorescence or phosphorescence. Examples of the target species include
a light-emitting substance that emits light by reaction with the binding substance,
and a light-emitting substance that emits light under excitation by the light supplied
from the light source 212. The present embodiment is constituted in such a manner
that the light detecting unit 213 can detect the light emitted (emission light).
[0294] With this constitution, in addition to the same advantages as are obtained in the
eleventh embodiment, the present embodiment offers the additional advantage that it
is possible to quantitatively and/or qualitatively analyze the sample using the detection
result of the emission light, analysis with higher precision therefore being realized.
Chemiluminescence such as fluorescence, in particular, is highly sensitive and is
therefore especially useful to detect minute reaction.
(P) Sixteenth Embodiment
[0295] Next, the sixteenth embodiment of the present invention is described with reference
to FIG. 24 and FIG. 25. In these figures, substantially identical components with
the eleventh through fifteenth embodiments are indicated by the same reference numerals.
[0296] The basic constitution of the present embodiment is identical with that of the eleventh
embodiment. Namely, the analysis apparatus is constituted in such a manner that the
light emitted from the light source 212 is reflected by the sensor chip 281 and detected
by the light detecting unit 213. Also, like the eleventh embodiment, the sensor chip
281 according to the present embodiment is identical with that of the conventional
sensor chip in its basic constitution. Namely, its surface (sensor surface) 281a is
covered with a metal layer 203, and a diffraction grating 205 is formed over plural
areas 251, 252, 253, 254, which are disposed on the metal layer 203.
[0297] As shown in FIG. 24, this embodiment has plural flow channels 280 through each of
which a sample flows, laid in a direction orthogonal to the direction in which the
diffraction grating 205 is formed. The flow channels 280 are disposed between a cover
286 covering the surface of the sensor chip 281 and the sensor chip 281, and are formed
in such a manner that they pass in pairs on each of the areas 251-254 on the sensor
chip. Interstices between one flow channel 280 and the other adjacent flow channel
280 are filled with seals such that the samples do not become mixed.
[0298] Moreover, as shown in FIG. 25, the present embodiment also has a sample introducing
apparatus 287, disposed upstream of the flow channels 280 of the analysis apparatus
210, for assigning different sample fluids to the flow channels 280, respectively,
and for introducing each of the assined sample fluids to the respective flow channel.
[0299] Because of these characteristics, when carrying out sample analysis method using
the sensor chip 281 of the present embodiment, in addition to the same steps as in
the eleventh embodiment, the method further has the step of assigning plural different
samples to the plural flow channels, respectively, and irradiating the sensor surface
with light while making each of the assigned samples flow through the respective flow
channel. Besides, it is also possible to carry out the same analysis as in the eleventh
embodiment for each of the flow channels.
[0300] With this constitution, in addition to the same advantages as are obtained in the
eleventh embodiment, the present embodiment also offers the advantage that since plural
target species can be analyzed at a time, it is possible to carry out analysis work
with efficiency. It further offers the additional advantage that since the plural
target species can be alanyzed under the same conditions, it is possible to analyze
differences between the target species clearly.
[0301] The above description will be further discussed in more detail.
[0302] An elevational view of the sensor chip 281 is shown in FIG. 26. As shown in FIG.
26(a), the diffraction grating 205 formed on the sensor chip 281 surface has different
groove pitches for each of the four areas (diffraction grating area) G1, G2, G3, G4,
which are disposed continuously in a direction perpendicular to the grooves.
[0303] On the sensor chip 281 surface, m flow channels P1, P2, ···, Pm are formed in a direction
perpendicular to the grooves. On the flow channels P1, P2, ···, Pm, the binding substance
206 is immobilized in spots for each of the areas G1, G2, G3, G4 having different
groove pitches.
[0304] In the following, advantages of the embodiment are explained in comparison with a
conventional example. In the conventional example shown in FIG. 26(b), a diffraction
grating with uniform groove pitches is formed on a sensor chip. If the sensor chip
is equipped with m flow channels P1, P2, ···, Pm, through each of which a sample flows
over the surface, the maximum number of target species which can be analyzed is m,
namely the same number as the number of the flow channels. However, the measurement
range of the conventional sensor chip is so narrow that when a resonance point falls
outside the measurement range upon light irradiation, or when the detection of more
resonance points is desired, it is necessary to alter the wavelength of the incident
light or to alter the incident angle.
[0305] On the othe hand, in the sensor chip 281 of the present invention, the maximum number
of target species which can be analyzed is still m, namely the same number as that
of the conventional example. Moreover, it is possible to detect a resonance point
without the need of altering the wavelength of the incident light or the incident
angle according to the groove pitch, and to detect resonance points spreading over
a wide measurement range according to the groove pitch.
[0306] As explained above, simply by forming both plural groove pitches and plural flow
channels, it is possible to obtain a sensor chip that offers the above-mentioned advantages.
Better still, by modifying the arrangement of the binding substance, it is possible
to obtain further advantages. In the following, explanation will be made in more detail
using examples. The constitution used in each of the following examples is identical
with that of the sixteenth embodiment mentioned above, except for the number of kinds
of areas and the combination of binding substances to be immobilized.
(Example 6-1)
[0307] In this example, explanation is made for the case where the angle of the reflected
light is detected while the incident angle of the incident light is varied.
[0308] As shown in FIG. 27(a), on a sensor chip 294 having two areas having different groove
pitches, namely, a low-angle detection part G1 and a high-angle detection part G2,
n kinds of binding substances S1, S2, ···, Sn are immobilized for each of the two
areas. At this point, to achieve the objective of analyzing the reaction between the
binding substances and the target species with a wide measurement range, the combination
of binding substances S1, S2, ···, Sn in each of the areas is determined to be identical
on the same flow channel.
[0309] Accordingly, m × n different kinds of combinations of a target species and a binding
substance can be detected, so that it becomes possible to analyze the number of target
species using the single sensor chip 294. Moreover, since measurement can be carried
out using two different measurement ranges of the low-angle detection part G1 and
the high-angle detection part G2, it is possible to carry out measurement with a wider
measurement range as a whole than that of the conventional method.
[0310] In the above case, incidentally, it is possible to determine voluntarily which binding
substance S1, S2, ···, Sn is immobilized in which area G1, G2 on which flow channel
P1, P2, ···, Pm, according to the target species to be analyzed.
(Example 6-2)
[0311] In this example, explanation is made for the case where the angle of the reflected
light is detected while the incident angle of the incident light is varied.
[0312] As shown in FIG. 27(b), on a sensor chip 295 having a number of areas G1, G2, ···,
Gi with different groove pitches, plural kinds of binding substances S1, S2, ···,
Sn are immobilized in n places for each of the areas G1, G2, ···, Gi.
[0313] With this arrangement, depending on the munber of the areas i, it becomes possible
to detect resonance points over a still wider measurement range. The kinds of binding
substances S1, S2, ···, Sn can be either unified over the areas G1, G2, ···, Gi, as
in the example 6-1, or varied independently of the areas. When the kinds of binding
substances S1, S2, ···, Sn are unified over the areas G1, G2, ···, Gi, it is possible
to measure plural reactions between the binding substance S1, S2, ···, Sn and the
target species with a wider measurement range, as in the example 6-1. On the other
hand, when the kinds of binding substance S1, S2, ···, Sn are varied between the areas
G1, G2, ···, Gi, it is possible to analyze plural reactions between the binding substances
S1, S2, ···, Sn, whose degrees of reaction are known, and the target species at a
time.
[0314] As is the case with the example 6-1, it is possible to determine voluntarily which
binding substance S1, S2, ···, Sn is immobilized in which area G1, G2, ···, Gi on
which flow channel P1, P2, ···, Pm, according to the target species to be analyzed.
(Example 6-3)
[0315] In this example, explanation is made for the case where the intensity of the reflection
light is detected while the incident angle of the incident light is fixed.
[0316] As shown in FIG. 27(c), on a sensor chip 296 having plural areas G1, G2, ···, Gi
with different groove pitches, n kinds of binding substances S1, S2, ···, Sn are immobilized
for each of the areas G1, G2, ···, Gi. At this point, to achieve the objective of
analyzing the reaction between the binding substances S1, S2, ···, Sn and the target
species with a wide measurement range, the combination of binding substances S1, S2,
···, Sn in each of the areas G1, G2, ···, Gi is determined to be identical on the
same flow channel.
[0317] With this arrangement, it is possible to analyze the reaction between the binding
substances S1, S2, ··· , Sn, which require a wide measurement range, and the target
species without the need of varying the incident angle of the incident light. Consequently,
in addition to being possible to analyze plural target species as in the example 6-1,
it is possible to carry out measurement with high time resolution since no time is
required to vary the incident angle of the incident light.
[0318] As is the case with the examples 6-1, 6-2, it is possible to determine voluntarily
which binding substance S1, S2, ···, Sn is immobilized in which area G1, G2, ···,
Gi on which flow channel P1, P2, ···P, according to the target species to be analyzed.
(Q) Seventeenth Embodiment
[0319] Next, the seventeenth embodiment of the present invention is described with reference
to FIG. 28, which is an enlarged view of the sensor chip surface. In this figure,
substantially identical components with the eleventh through sixteenth embodiments
are indicated by the same reference numerals.
[0320] The present embodiment has a structure using the same analysis apparatus 210 and
the same sensor chip as those of the eleventh embodiment.
[0321] In this embodiment, as shown in FIG. 28(a), the spots of the binding substances 206,
207 are immobilized so as to be across boundaries between the areas 251, 252 and 253,
254, which have different groove pitches.
[0322] With this arrangement, it is possible to carry out analysis with a small number of
spots in which the binding substances 206, 207 are immobilized. By extension, it becomes
possible to miniaturize the sensor chip and to increase the number of reactions that
can be detected with the single detection.
[0323] The above description will be further discussed in more detail.
[0324] In the conventional example shown in FIG. 28(b), each of the spots of the binding
substances 206, 207 are immobilized within one of the areas 251-254. In this case,
it is necessary to immobilize the same number of spots as the number areas 251-254
for each of the binding substances 206, 207, and only a single detection area 293
can be disposed for each of the spots. Incidentially, the detection area 293 means
an area by which the reflection light for detection is reflected. Although having
a rectangular shape in FIG. 28, the detection area 293 may also have any other arbitrarily
determined shape such as, for example, a rounder shape.
[0325] On the other hand, as shown in FIG. 28(a), by immobilizing the spots of binding substances
206, 207 so as to be across boundaries between areas 251, 252 and 253, 254 with different
groove pitches, it becomes possible to divide each of the spots into two detection
areas between one area and another area having different groove pitches. Consequently,
it is possible to carry out measurement with two different measurement ranges at a
single spot.
[0326] The position in which a binding substance is immobilized is not limited to the boundary
between two areas having different groove pitches, but also can be the boundary between
more than two areas as long as the areas having different groove pitches. FIG. 28(c)
shows the case where a diffraction grating is formed on a sensor chip in such a manner
that four areas 251-254 with different groove pitches define a cross-shaped boundary.
In this case, by immobilizing each spot of the binding substances 206, 207 so as to
enclose the center of the boundary's cross shape, it is possible to use each of the
overlapping areas between the individual spot of the binding substances 206, 207 and
each of the areas 251-254 as a detection area 293, namely, it is possible to make
four detection areas 293 at a single spot. Consequently, it is possible to carry out
measurement with four different measurement ranges at a single spot.
(R) Eighteenth Embodiment
[0327] Next, the eighteenth embodiment of the present invention is described with reference
to FIGs. 25, 29. In these figures, substantially identical components with the eleventh
through seventeenth embodiment are indicated by the same reference numerals.
[0328] The basic constitution of the present embodiment is identical with that of the eleventh
embodiment. Namely, the analysis apparatus is constituted in such a manner that the
light emitted from the light source 212 is reflected by the sensor chip 300 and detected
by the light detecting unit 213. Also, like the eleventh embodiment, the sensor chip
300 according to the present embodiment is identical with that of the conventional
sensor chip in its basic constitution. Namely, its surface (sensor surface) 281a is
covered with a metal layer 203, and a diffraction grating 205 is formed over the metal
layer 203 over plural areas 251, 252, 253, 254, which are disposed on the metal layer
203.
[0329] As shown in FIG. 29, this embodiment has plural flow channels 280, through each of
which a sample flows, laid in a direction orthogonal to an uneven surface being a
diffraction grating 205. The flow channels 280 are disposed between a cover 286 covering
the surface of the sensor chip 300 and the sensor chip 300, and are formed in such
a manner that they pass together on each of the areas 251-254 on the sensor chip.
Interstices between these two adjacent flow channels 280 are filled with seals such
that the samples do not become mixed.
[0330] In each of the areas 251-254 on the flow channels 280, a binding substance 206 and
a non-binding substance 291 are imobilized. The non-binding substance 291 is a substance
that does not have the properties of binding specifically to the target species to
be detected, and the appropriate non-binding substance 291 is selected in accordance
with the target species (chemical species, biochemical species, biological species,
etc.) to be detected. When the sample contains plural target species, plural binding
substances 206 and plural non-binding substances 291 are selected according to the
plural target species, respectively, and immobilized on different areas 251-254. Consequently,
the area on which the binding substance 206 is immobilized functions as a reaction
area, while the area on which the non-binding substance 291 is immobilized and the
area on which either the binding substance 206 or the non-binding substance 291 is
not immobilized each function as a non-reaction area. If the metal layer 203 is composed
of a metal that does not bind specifically to the target species, it is also preferable
that any non-binding substance 2091 is not immobilized, and the area on which either
the binding substance 206 or the non-binding substance 291 is not immobilized and
in which the metal layer 203 is laid bare is used as a non-reaction area.
[0331] Moreover, as shown in FIG. 25, the present embodiment also has a sample introducing
apparatus 287, disposed upstream of the flow channels 280 of the analysis apparatus
210, for assigning different sample fluids to the flow channels 280, respectively,
and for introducing each of the assined sample fluids to the respective flow channel.
[0332] The remaining constitution is identical with that of the eleventh embodiment.
[0333] With this structure, the reflection light from the individual area 251-254 includes
the light that has been reflected by the reaction area and the light that has been
reflected by the non-reaction area. The reflection light reflected by the reaction
area changes according to quantitative and/or qualitative factors in connection with
the sample, while the reflection light reflected by the non-reaction area is not affected
by the sample and its intensity is determined only by the structure of the diffraction
grating surface.
[0334] Because of these characteristics, when carrying out sample analysis method using
the sensor chip 300 of the present embodiment, in addition the same steps as in the
eleventh embodiment, the method further has the step of assigning plural different
samples to the plural flow channels, respectively, and irradiating the sensor surface
with light while making each of the assigned samples flow through the respective flow
channel, the step of identifying a groove pitch with which a surface plasmon resonance
occurs for each of the flow channels and for each of the reaction area and the non-reaction
area, and the step of analyzing based on the groove pitch of the reaction area and
the non-reaction area for each of the flow channels.
[0335] On this account, since the reflection light from a reaction area where the binding
substance binds to the target species can be analyzed using the reflection light from
the non-reaction area adjacent to the reaction area as a reference, in addition to
the same advantages as are obtained by the eleventh embodiment, it is possible to
analyze reliably the change caused by the specific binding of the binding substance
to the target species, as in the thirteenth embodiment. Besides, as in the sixteenth
embodiment, since plural samples can be analyzed at a time, it is possible to carry
out analysis work with efficiency. Also, since the plural sample can be alanyzed under
the same conditions, it is possible to analyze differences between the samples clearly.
(S) Nineteenth Embodiment
[0336] Next, the constitution of a sensor chip (surface plasmon resonance sensor chip) 221
according to the nineteenth embodiment of the present invention is described with
reference to FIG. 30.
[0337] As is the eleventh embodiment, the sensor chip 221 according to the present embodiment
is identical with the conventional sensor chip in its basic constitution. Namely,
its surface (sensor surface) 221a is covered with a metal layer 223, and a diffraction
grating 225 is formed on the metal layer 223. In the present embodiment, the diffraction
grating 225 is formed in such a manner that its groove pitches have a continuous distribution.
Specifically, FIG. 31 shows groove pitches viewed in a direction of the arrow X in
FIG. 30. As indicated by the solid line in FIG. 31, a diffraction grating 225 is formed
in such a manner that the groove pitches vary smoothly between two each adjacent grooves.
Incidentally, in FIG. 31, the single-dashed line indicates the variation of groove
pitches in the sensor chip of the eleventh embodiment, while the double-dashed line
indicates the variation of groove pitches in the conventional sensor chip.
[0338] The wave number of the evanescent wave occurs along the diffraction grating 225 upon
light irradiation depends on, as indicated by the formula 2, the grating constant
of the diffraction grating 225, which grating constant depends on the groove pitches
of the diffraction grating 225. Consequently, with the above structure, by using irradiation
light with a constant wave number, it is possible to obtain various evanescent waves
whose wave numbers vary in a continuous distribution. FIG. 32 is a dispersion relationship
diagram in this case, showing the relation between the angular frequency ω and the
wave number k for each of the surface plasmon wave and the evanescent wave.
[0339] In FIG. 32, the band indicated by the reference code C shows the relation between
the angular frequencies ω and the wave numbers k of evanescent waves that occur along
all of the diffraction grating 225 as a whole. The band is an aggregation of straight
lines parallel to the straight line B0, which indicates the relation between the angular
frequency ω and the wave number k of the incident light. In the figure, the ranges
within which the curved lines A1, A2 overlap with the band C are indicated by the
line segments S1-S2, S3-S4, which also indicate severally the ranges within which
a surface plasmon resonance occurs.
[0340] Since the ranges within which a surface plasmon resonance spread seamlessly as described
above, even when irradiating with light of a single wavelength at a predetermined
incident angle, a resonance phenomenon occurs at least in any positions on the diffraction
grating 225. In FIG. 32, for example, when irradiating with the light whose angular
frequency is ωx, the point Px of the angular frequency ωx on the curved line A1 is
a resonance point at which a resonance phenomenon occurs. An evanescent wave (indicated
by the straight line Bx) that passes through the resonance point Px is the evanescent
wave that resonates with the surface plasmon wave indicated by the curved line A1.
On the diffraction grating 225, a position that has the groove pitch corresponding
to the straight line Bx is assumed as the position in which a resonance phenomenon
actually occurs.
[0341] With the sensor chip 221 according to the present embodiment, since a resonance phenomenon
occurs at least in any position on the diffraction grating 225, in order to identify
the groove pitch with which a resonance phenomenon occurs, it is unnecessary to carry
out calculation such as approximation or interpolation, as is needed in the eleventh
embodiment. Consequently, when using the analysis apparatus of FIG. 20, sample analysis
can be carried out simply by detecting a position in which a resonance phenomenon
occurs from the intensity of the reflection light, and by comparing the groove pitch
corresponding to the position (in addition to the resonance wavelength determinatied
from the reflection-light intensity, in the case of a wavelength-change type, or the
resonance angle determined from the reflection-light intensity, in the case of an
angle-change type) with a working curve. It becomes therefore possible to analyze
the concentration of target species in the sample more easily and more precisely.
(T) Twentieth Embodiment
[0342] Next, the constitution of a sensor chip (surface plasmon resonance sensor chip) 231
according to the twentieth embodiment of the present invention is described with reference
to FIG. 33.
[0343] Like the conventional sensor chip, the sensor chip 231 according to the present embodiment
is constituted in such a manner that its surface (sensor surface) 231a is covered
with a metal layer 233, and that a diffraction grating 235 is formed on the metal
layer 233. Besides, in the present embodiment, plural determination areas 238a-238d
are disposed discretely on the metal layer 233, and a diffraction grating 235 is formed
in each of the determination areas 238a-238d.
[0344] Among the diffraction gratings 235 formed in the determination areas 238a-238d, respectively,
their groove pitches are identical with each other while their groove orientations
are different from each other. Specifically, when the groove orientation of the diffraction
grating 235 in the determination area 238a is used as a reference, the groove orientation
in the determination area 238b is inclined θ2, the groove orientation in the determination
area 238c is inclined θ3, and the groove orientation in the determination area 238d
is inclined θ4 toward the reference. As a result, when viewed from a direction perpendicular
to the groove orientation in the determination area 238a, a substantial groove pitch
in the determination area 238a is still d1, while a substantial groove pitch d2 in
the determination area 238b is d1/cosθ2, a substantial groove pitch d3 in the determination
area 238c is dl/cosθ3, and a substantial groove pitch d4 in the determination area
238d is d1/cosθ4. When the relation between the inclination angles is assumed as θ2
< θ3 < θ4, the relation between the substantial groove pitches is defined as d1 <
d2 < d3 < d4. The substantial groove pitches therefore differ among the determination
areas 238a-238d.
[0345] Consequently, when the sensor chip 231 according to the present embodiment is irradiated
with light in a specific direction, each of the determination areas 238a-238d produces
an evanescent wave whose wave number in accordance with a substantial groove pitch
when respect to the irradiation direction. Accordingly, resonance points at each of
which a surface plasmon wave resonates with an evanescent wave also have a certain
distribution. Consequently, as in the eleventh embodiment, it is possible to analyze
a sample having a wide refractive index distribution even if the measurement range
is limited.
(U) Others
[0346] Up to this point, several embodiments of the present invention have been explained,
although the present invention is not limited by the above embodiments but also can
be carried out with various modifications as long as it departs from the gist of the
present invention.
[0347] While in the first embodiment, for Example, the center diffraction grating surface
5e is disposed parallel to the reference plane S0, it is also possible to adopt an
arrangement as shown in FIG. 34. In the figure, diffraction grating surfaces 416a-416e
are arranged in such a manner that a diffraction grating surface 416e at one end is
disposed parallel to the reference plane S0, while the inclination angles of the remaining
diffraction grating surfaces 416a-416d with respect to a light irradiation diretion
gradually increase in turn. However, when disposing a definite number of diffraction
grating surfaces, it is preferable to adopt an arrangement as in the first embodiment,
namely, the inclination angles gradually increase from the center diffraction grating
surface 5e toward either side. With this arrangement, it is possible to make prominence
of the center relatively low so as not to prevent the flow of a sample fluid.
[0348] Further, it is not necessarily required to arrange the diffraction grating surfaces
continuously. It is also possible to dispose the individual diffraction grating surface
426a-426e as shown in FIG. 35 in such a manner that one end is disposed in the same
level as the reference plane S0 while the other end is raised so that it forms a certain
inclination angle with respect to the reference plane S0.
[0349] In order to dispose a larger number of diffraction grating surfaces in a single measurement
spot, it is also possible to arrange the diffraction grating surfaces 436a-436k in
plural lines as shown in FIG. 36. With this arrangement, it becomes possible to dispose
a number of diffraction grating surfaces 436a-436k in a compact size.
[0350] Among the above embodiments, some embodiments dispose planate diffraction grating
surfaces 5a-5i in such a manner that when viewed from a diretion parallel to the specific
plane S1, the inclination angles with respect to the reference plane S0, while other
embodiments use a diffraction grating surface 25 having a curved surface whose light-irradiation
side bulges out. These arrangements and shapes are intended for preventing the rays
of light reflected by the diffraction grating surfaces 5a-5i or the rays of light
reflected by various positions on the diffraction grating surface 25 from being mixed
with each other. However, the present invention is not limited to these arrangements
or shapes as long as it is possible to identify the reflection light from the individual
diffraction grating surface 5a-5i or from the individual position on the diffraction
grating surface 25.
[0351] Further, in the above embodiments, the sensor chip of the present invention is constituted
as a sensor chip for multipoint simultaneous measurement purpose (or multi-item measurement
purpose), in which plural measurement spots are disposed on the sensor surface. However,
it is also possible to apply the present invention to a sensor chip whose sensor surface
serves as a single measurement spot as a whole.
[0352] While the above embodiments refer to the case where the present invention is applied
to a conventional diffraction-grating type sensor chip having a general structure,
in which the diffraction grating is formed on the surface of the metal layer, the
present invention is also applicable to various diffraction-grating type sensor chips
having other structures. Specifically, it is possible to apply the present invention
to any sensor chip as long as it has a diffraction grating surface on which a diffraction
grating is formed, which diffraction grating generates an evanescent wave upon light
irradiation, and a metal layer that is disposed along the diffraction grating surface
and along which surface a surface plasmon wave can be induced upon light irradiation.
[0353] It is also possible to carry out two or more of the above embodiments in combination.
Specifically, it is preferable to carry out, for example, the third embodiment and
the sixth embodiment in combination, as well as the fifth embodiment and the seventh
embodiment in combination.
[0354] It is especially preferable to combine the third embodiment and/or the eighth embodiment
with the fourth embodiment and/or the ninth embodiment to carry out both the correction
using a non-diffraction surface and the correction using a non-reaction area, because
it enables to carry out analysis reliably with high precision. Specifically, the light
reflected from a reaction area is corrected based on a non-diffraction surface to
thereby calculate a resonance angle while the light reflected from a non-reaction
area is corrected based on a non-diffraction surface to thereby calculate another
resonance angle. Based on both of the resonance angles, it is possible to calculate
the shift amount of resonance angle resulting from a real specific reaction to thereby
obtain the concentration of a target species.
[0355] In the fourth embodiment and the ninth embodiment, there are two cases regarding
the selection of a non-binding substance: the case where a substance not having the
property of causing any specific reaction with a target species is selected in accordance
with the target species; and the case where the same substance is used regardless
of the target species.
[0356] In the case where an appropriate substance is selected in accordance with the target
species, it is preferable to immobilize both the binding substance and the non-binding
substance in the same diffraction grating surface, as in the fourth embodiment. Considering
labor savings in production of the sensor chip, on the contrary, it is more advantageous
to immobilize a non-binding substance on a measurement spot adjacent to a measurement
spot on which the binding substance is immobilized so as to use the adjacent measurement
spot as a non-reaction area, as in the ninth embodiment.
[0357] On the other hand, in the case where the same substance is used as a non-reaction
substance for all of the target species, it is possible to use BSA (bovine serum albumin),
which is used as a blocking agent, or gelatin. In this case, it is advantageous in
view of manufacturability to immobilize a non-reaction substance on all of the diffraction
grating surfaces in one or more of the measurement spots on the sensor chip and to
use these one or more measurement spots as non-reaction areas, as in the ninth embodiment.
[0358] Meanwhile, in the modification of the eleventh embodiment (refer to FIG. 19), the
areas 251-254 are disposed discretely in such a manner that those groove orientations
are uniform. On the contrary, it is also preferable to dispose the areas 251-53 as
the sensor chip 1" shown in FIG. 37, namely, in such a manner that the groove orientation
of one or more of the areas 254 is different from those of the remaining areas 251-53,
or in such a manner that the groove orientations of all of the areas 251-254 are different
with each other. In this case, by light irradiating while varying a light irradiation
diretion in accordance with the areas 251-254 such that the light enters perpendicular
to the groove orientation of each area, it is possible to achieve the same operations
and advantages as are achieved in the eleventh embodiment.
[0359] Besides, as shown in FIG. 38, by preparing plural sensor chips 241A-241D with different
groove pitches to use the aggregate of these sensor chips as a single sensor chip
241, it is possible to achieve the same operations and advantages as are achieved
in the eleventh embodiment.
[0360] While the above embodiments refer to the case of using a conventional diffraction-grating
type sensor chip having a general structure, the present invention can be also carried
out using various diffraction-grating type sensor chip having other structures. Specifically,
it is possible to apply the present invention to any sensor chip as long as it has
a sensor surface in contact with the sample, a metal layer that is disposed in the
vicinity of the sesor surface and on which a surface plasmon wave can be induced and
a diffraction grating that is disposed in the vicinity of the sensor surface and generates
an evanescent wave upon light irradiation.
[0361] The above embodiments does not necessarily require to be carried out one severally,
but also can be carried out two or more in combination.
[0362] Besides, the kind of binding substance to be immobilized on each of the areas with
different groove pitches can be determined arbitrarily according to need.
[0363] It is also possible to carry out two or more of the above embodiments in combination.
Specifically, it is preferable to carry out, for example, the twelfth embodiment and
the fourteenth embodiment in combination.
[0364] It is especially preferable to combine the twelfth embodiment with the thirteenth
embodiment to carry out both the correction using a non-diffraction surface and the
correction using a non-reaction area, because it enables to carry out analysis reliably
with high precision. Specifically, the light reflected from a reaction area is corrected
based on a non-diffraction surface to thereby calculate a resonance angle while the
light reflected from a non-reaction area is corrected based on a non-diffraction surface
to thereby calculate another resonance angle. Based on both of the resonance angles,
it is possible to calculate the shift amount of resonance angle resulting from a real
specific reaction to thereby obtain the concentration of a target species.
[0365] In the thirteenth embodiment, there are two cases regarding the selection of a non-binding
substance: the case where a substance not having the property of causing any specific
reaction with a target species is selected in accordance with the target species;
and the case where the same substance is used regardless of the target species.
[0366] In the case where the same substance is used as a non-reaction substance for all
of the target species, it is possible to use BSA (bovine serum albumin), which is
used as a blocking agent, or gelatin.
[0367] As in the examples given above, the present invention can be carried out with various
modifications as long as it departs from the gist of the present invention.
[0368] Description will be made hereinbelow of working examples. It is to be noted that
the present invention should by no means be limited to the following examples, and
various changes or modifications may be suggested without departing from the gist
of the invention.
[Example 1]
[0369] On the surface of a flat-shaped substrate of polycarbonate, there were provided uneven
forms having projections and depressions with a groove pitch of approximately 870
nm and a groove depth of approximately 40 nm as diffraction gratings. A depth of approximately
80 nm of gold was then evaporated onto the surface of the substrate, thus producing
a sensor chip.
[0370] After that, this sensor chip was formed into a rectangular shape with side-lengths
of 15 mm and 25 mm such that the diffraction gratings were arranged thereon in parallel
to the 25-mm sides. FIG. 39 shows an example of the thus produced sensor chip.
[0371] Subsequently, this sensor chip was folded approximately 0.8° on two fold lines each
of which was parallel to a 25-mm side of the sensor chip and which included points
locating on a 15-mm side, approximately 5.5 mm, 4mm, and 5.5 mm, respectively, from
one of its edges. More precisely, as shown in FIG. 40, a 5.5 mm × 25 mm rectangular
area (hereinafter called area A) and a 4 mm × 25 mm rectangular area (hereinafter
called area B) were adjacent to each other, in contact with each other on one of their
25-mm sides. On the other side of the area B, opposing to the area A with respect
to the area B, there was another 5.5 mm × 25 mm rectangular area (hereinafter called
area C) that was in contact with the area B on one of their 25-mm sides. The chip
was folded in such a way that the area B formed an angle of 0° with the reference
plane; that the area C formed an angle of approximately 0.8° with the area B; and
that the area A formed an angle of approximately -0.8° with the area B. Accordingly,
the area A formed an angle of approximately 1.6° with the area C. Further, the diffraction
grating was placed on the convex side of the folded sensor chip, so that a sample
could be assessed on the convex side of the sensor chip.
[0372] A central 10 mm × 10 mm area of the thus produced sensor chip served as a measurement
spot, and angle scanning was performed using a resonance angle detecting type SPR
assessment device, FLEX CHIPS™ Kinetic Analysis System (
HTS Biosystems Inc.), to measure the intensity of reflected light. With use of light of a wavelength
of approximately 870 nm as incident light, and purified water, as a sample, measurement
was performed at arbitrary three points in each of the above-mentioned areas.
[0373] FIG. 41 is a plot showing relations between the incident angle of incident light
and the intensity of reflected light measured, in the neighborhood of the resonance
angles. In each of the graphs, an angle at which the reflected light intensity takes
a minimum value indicates a resonance angle. Table 1 shows measurement results in
each of the areas, where SD denotes the standard variation; CV, the coefficient of
variation.
[Table 1]
Area |
Point |
Resonance angle of purified water |
Average |
SD |
CV(%) |
A |
A-1 |
21.616 |
|
|
|
|
A-2 |
21.645 |
|
|
|
|
A-3 |
21.663 |
21.641 |
0.024 |
0.111 |
B |
B-1 |
20.802 |
|
|
|
|
B-2 |
20.789 |
|
|
|
|
B-3 |
20.781 |
20.791 |
0.011 |
0.051 |
C |
C-1 |
19.818 |
|
|
|
|
C-2 |
19.870 |
|
|
|
|
C-3 |
19.891 |
19.860 |
0.038 |
0.189 |
[0374] The three measurement points in the area A were designated as A-1, A-2, and A-3,
respectively; the three measurement points in the area B were designated as B-1, B-2,
and B-3, respectively; and three measurement points in the area C were designated
as C-1, C-2, and C-3, respectively;
[0375] As shown in Table 1, the average resonance angles in the areas A, B, and C were 21.641°,
20.791°, and 19,860°, respectively.
[0376] Next, a flat-shaped sensor chip was separately prepared without being folded as in
FIG. 39, and angle scanning was performed, as in the cases of the areas A, B, and
C, using a resonance angle detecting type SPR assessment device, FLEX CHIPS™ Kinetic
Analysis System, to measure the intensity of reflected light. With use of a light
beam of a wavelength of approximately 870 nm as incident light, and purified water,
as a sample, measurement was performed at 400 points in area D of the flat-shaped
sensor chip.
[0377] FIG. 42 is a plot showing relations measured between the incident angle of the incident
light and the intensity of reflected light, in the neighborhood of the resonance angle.
Table 2 shows measurement results.
[Table 2]
Area |
The number of points |
Resonance angle of purified water |
Average |
SD |
CV(%) |
D |
400 |
Maximum 20.877 |
20.832 |
0.019 |
0.092 |
|
|
Minimum 20.797 |
|
|
|
[0378] As shown in FIG. 42, all the measurement points in the area D reveal one identical
resonance angle, and the average resonance angle is 20.832° as shown in FIG. 2.
[0379] Partly since the diffraction gratings in the areas A, B, C, and D had the same groove
pitch and the same groove depth, and partly since the same sample, purified water,
was used in the measurement, the resonance angles of these areas should have taken
one identical value. However, since the areas A, B, and C had an angle of -0.809°,
0.041°, and 0.972°, respectively, in comparison with the area D for which no angle
was introduced, the measured resonance angles revealed apparently different values.
[0380] These results indicate that it is possible to vary the incident angle of the incident
light that enters a diffraction grating surface, by inclining the diffraction grating
surface to have an inclination angle.
[Example 2]
[0381] As in the case of Example 1, on the surface of a flat-shaped substrate of polycarbonate,
there were provided diffraction gratings formed of projections and depressions having
a groove pitch of approximately 870 nm and a groove depth of approximately 40 nm.
A depth of approximately 80 nm of gold was then evaporated onto the surface of the
substrate, thus producing a sensor chip. This sensor chip was folded to have 26 surfaces
in such a manner that each of the surfaces had a different inclination angle. Hereinafter,
the areas of those surfaces with such different inclination angles were designated
as area NO. 1, area NO. 2, and so forth, giving them natural numbers in an ascending
order of the inclination angle formed by each area with a horizontal surface.
[0382] Using the thus produced sensor chip that was given 26 surfaces with different inclination
angles, angle scanning was performed by an SPR assessment device, FLEX CHIPS™ Kinetic
Analysis System, to measure the intensity of reflected light. The sample was purified
water, and a light beam of a wavelength of approximately 870 nm was used as incident
light. The resonance angle of each of the areas was identified to compare with the
measurement result in the area D, which had no inclination angle, in Example 1, so
as to find the inclination angles of the separate areas. The following table 3 shows
the measurement results.
[Table 3]
Area NO. |
Resonance angle of purified water |
Angle of the diffraction grating surface |
1 |
21.768 |
-0.936 |
2 |
21.746 |
-0.914 |
3 |
21.734 |
-0.902 |
4 |
21.713 |
-0.881 |
5 |
21.697 |
-0.865 |
6 |
21.665 |
-0.833 |
7 |
21.572 |
-0.740 |
8 |
21.415 |
-0.583 |
9 |
21.364 |
-0.532 |
10 |
21.263 |
-0.431 |
11 |
21.164 |
-0.332 |
12 |
21.120 |
-0.288 |
13 |
21.053 |
-0.221 |
14 |
21.008 |
-0.176 |
15 |
20.954 |
-0.122 |
16 |
20.915 |
-0.083 |
17 |
20.864 |
-0.032 |
18 |
20.802 |
0.030 |
19 |
20.703 |
0.129 |
20 |
20.522 |
0.310 |
21 |
20.284 |
0.548 |
22 |
20.095 |
0.737 |
23 |
20.028 |
0.804 |
24 |
19.971 |
0.861 |
25 |
19.907 |
0.925 |
26 |
19.854 |
0.978 |
[0383] As a result, it was found that the areas had separate angles ranging from -0.936°
to 0.978°.
[0384] After that, using this sensor chip in an SPR assessment device, FLEX CHIPS™ Kinetic
Analysis System, the intensity of reflected light was measured. A light beam of a
wavelength of approximately 870 nm was used as incident light, and ethanol aqueous
solution concentrations of 2.5, 5, 10, 20, 30, 40, and 50 per cent were used as samples.
[0385] The following table 4 shows the concentration of ethanol in each area and the amount
(hereinafter called a "shift amount") of change in the resonance angle caused by increasing
the ethanol concentration.
[Table 4]
Area No. |
The shift amount of the resonance angle (in degrees) |
|
EtOH 0% |
2.50% |
5% |
10% |
20% |
30% |
40% |
50% |
1 |
0.0000 |
0.0714 |
0.1367 |
0.2763 |
0.5883 |
0.9407 |
Exceeding |
Exceeding |
|
|
|
|
|
|
|
the range |
the range |
2 |
0.0000 |
0.0698 |
0.1359 |
0.2783 |
0.5934 |
0.9519 |
Exceeding |
Exceeding |
|
|
|
|
|
|
|
the range |
the range |
3 |
0.0000 |
0.0726 |
0.1412 |
0.2907 |
0.6005 |
0.9487 |
Exceeding |
Exceeding |
|
|
|
|
|
|
|
the range |
the range |
4 |
0.0000 |
0.0714 |
0.1373 |
0.2802 |
0.5898 |
0.9455 |
1.1919 |
Exceeding |
|
|
|
|
|
|
|
|
the range |
5 |
0.0000 |
0.0740 |
0.1418 |
0.2850 |
0.5988 |
0.9487 |
1.1865 |
Exceeding |
|
|
|
|
|
|
|
|
the range |
6 |
0.0000 |
0.0696 |
0.1366 |
0.2798 |
0.5911 |
0.9326 |
1.1738 |
Exceeding |
|
|
|
|
|
|
|
|
the range |
7 |
0.0000 |
0.0723 |
0.1409 |
0.2887 |
0.5956 |
0.9498 |
1.1771 |
Exceeding |
|
|
|
|
|
|
|
|
the range |
8 |
0.0000 |
0.0735 |
0.1441 |
0.2878 |
0.5963 |
0.9511 |
1.1844 |
1.4028 |
9 |
0.0000 |
0.0822 |
0.1679 |
0.3460 |
0.6277 |
0.9709 |
1.2037 |
1.4343 |
10 |
0.0000 |
0.0731 |
0.1420 |
0.2817 |
0.5960 |
0.9570 |
1.1932 |
1.4352 |
11 |
0.0000 |
0.0690 |
0.1373 |
0.2816 |
0.6012 |
0.9669 |
1.2186 |
1.4489 |
12 |
0.0000 |
0.0727 |
0.1381 |
0.2756 |
0.5912 |
0.9207 |
1.1873 |
1.4777 |
13 |
0.0000 |
0.0730 |
0.1406 |
0.2899 |
0.6178 |
0.9692 |
1.2071 |
1.4075 |
14 |
0.0000 |
0.0774 |
0.1546 |
0.3095 |
0.6228 |
0.9874 |
1.2217 |
1.4492 |
15 |
0.0000 |
0.0744 |
0.1459 |
0.2880 |
0.6114 |
0.9601 |
1.1994 |
1.4247 |
16 |
0.0000 |
0.0727 |
0.1401 |
0.2842 |
0.6216 |
0.9583 |
1.1994 |
1.4241 |
17 |
0.0000 |
0.0755 |
0.1454 |
0.2950 |
0.6290 |
0.9650 |
1.2042 |
1.4334 |
18 |
0.0000 |
0.0726 |
0.1484 |
0.3111 |
0.6276 |
0.9698 |
1.2120 |
1.4377 |
19 |
0.0000 |
0.0741 |
0.1520 |
0.3161 |
0.6290 |
0.9731 |
1.2131 |
1.4418 |
20 |
0.0000 |
0.0803 |
0.1639 |
0.3240 |
0.6252 |
0.9799 |
1.2157 |
1.4526 |
21 |
0.0000 |
0.0715 |
0.1488 |
0.3185 |
0.6316 |
0.9759 |
1.2106 |
1.4484 |
22 |
0.0000 |
0.0771 |
0.1585 |
0.3337 |
0.6417 |
0.9763 |
1.2204 |
1.4552 |
23 |
0.0000 |
0.0862 |
0.1639 |
0.3051 |
0.6255 |
0.9574 |
1.1977 |
1.4272 |
24 |
0.0000 |
0.0720 |
0.1434 |
0.2930 |
0.6112 |
0.9464 |
1.1845 |
1.4189 |
25 |
0.0000 |
0.0723 |
0.1439 |
0.2980 |
0.6232 |
0.9536 |
1.1885 |
1.4311 |
26 |
0.0000 |
0.0764 |
0.1529 |
0.3034 |
0.6236 |
0.9573 |
1.1970 |
1.4381 |
Average |
|
0.0741 |
0.1462 |
0.2970 |
0.6120 |
0.9582 |
1.1995 |
1.4362 |
SD |
|
0.0039 |
0.0092 |
0.0187 |
0.0163 |
0.0152 |
0.0139 |
0.0175 |
CV(%) |
|
5.30 |
6.27 |
6.28 |
2.67 |
1.58 |
1.16 |
1.22 |
[0386] In all the areas, the coefficient of variation (CV) at each the ethanol concentration
takes favorable values ranging from 1.2 to 6.3 per cent. In addition, the shift amount
of the resonance angle in each area reveals the same value.
[0387] In consequence, it is apparent that the measurement accuracy is not affected even
if a diffraction grating surface is inclined.
[Example 3]
[0388] Of the measurement data obtained in the above Example 2, measurement data obtained
when the incident angle of incident light was 21.5° was selected, and calculation
was performed within this selected data to obtain the angle of the area that reveals
a minimal intensity of reflected light when ethanol aqueous solution concentrations
of 0, 2.5, 5, 10, 20, 30, 40, and 50 per cent, respectively, were used as samples.
More precisely, the angle of each area (Table 3) and the intensity of reflected light
in the area were used in regression calculation of an angle at which the intensity
of reflected light takes a minimal value.
[0389] FIG. 43 shows relations between the concentration of ethanol aqueous solution and
the shift amount of the angle (or the resonance angle) of the area that reveals a
minimal intensity of reflected light.
[0390] On the basis of the resonance angle obtained with a generally practiced technique,
or angle scanning, relationship between the concentration of ethanol aqueous solution
and the shift amount of the resonance angle of the diffraction grating surfaces on
areas NO. 7 and NO. 15, was examined. FIG. 44 shows such examination results.
[0391] The following table 5 shows the comparison result between FIG. 43 and FIG. 44.
[Table 5]
Ethanol concentration |
Diffraction grating surface with a minimal intensity of reflected light |
Area NO. 7 |
Area NO. 15 |
|
Angle |
Shift amount |
Resonance angle |
Shift amount |
Resonance angle |
Shift amount |
0% |
-0.723 |
0.000 |
21.572 |
0.000 |
20.954 |
0.000 |
2.50% |
-0.622 |
0.101 |
21.644 |
0.072 |
21.029 |
0.075 |
5% |
-0.539 |
0.184 |
21.712 |
0.140 |
21.100 |
0.146 |
10% |
-0.382 |
0.341 |
21.860 |
0.288 |
21.242 |
0.288 |
20% |
-0.032 |
0.690 |
22.167 |
0.595 |
21.565 |
0.611 |
30% |
0.331 |
1.053 |
22.521 |
0.949 |
21.914 |
0.960 |
40% |
0.533 |
1.256 |
22.749 |
1.177 |
22.153 |
1.199 |
50% |
0.742 |
1.465 |
|
|
22.379 |
1.425 |
[0392] As a comparison result, it was found that FIG. 43 and FIG. 44 show similar shift
amounts for each concentration of ethanol. Therefore, utilizing a sensor chip provided
with diffraction grating surfaces according to the present invention, it is possible
to obtain the shift amount of the resonance angle, which has been found as a result
of a generally performed technique (FIG. 44), in the form of a variation in the resonance
angle revealed by the diffraction grating surface that reveals the minimal reflection
intensity, by using a measurement method in which the incident angle of incident light
is not changed.
[Example 4]
[0393] Of the measurement data obtained in Example 2, data analysis was performed on the
data that was obtained when the incident angle of incident light was 21.5° or 21.0°.
[0394] In a case where the incident angle of incident light was 21.5°, relation between
the intensity of reflected light and ethanol concentrations was analyzed in areas
NO. 2, NO. 7, NO. 10, NO. 16, and NO. 19. FIG. 45 and table 6 show the analysis result.
In a case where the incident angle of incident light was 21.0°, relation between the
intensity of reflected light and ethanol concentration was analyzed in areas NO. 10,
NO. 13, NO. 18, NO. 20, and NO. 22. FIG. 46 and table 7 show the analysis result.
[Table 6]
An incident angle of 21.5° |
Ethanol concentration |
Diffraction grating surface area |
|
No.2 |
No.7 |
No.10 |
No.16 |
No.19 |
0% |
1278.07 |
|
|
|
|
2.50% |
1461.97 |
1101.32 |
|
|
|
5% |
1648.93 |
1223.44 |
|
|
|
10% |
2026.36 |
1590.83 |
1039.49 |
|
|
20% |
2541.48 |
2314.61 |
1580.55 |
1015.67 |
|
30% |
2789.89 |
2707.64 |
2369.13 |
1593.10 |
1078.98 |
40% |
|
2825.09 |
2644.40 |
2158.68 |
1592.99 |
50% |
|
|
2767.90 |
2484.18 |
2068.05 |
[Table 7]
An incident angle of 21.0° |
Ethanol concentration |
Diffraction grating surface area |
|
No.10 |
No.13 |
No.18 |
No.20 |
No.22 |
0% |
1353.38 |
|
|
|
|
2.50% |
1543.41 |
1049.68 |
|
|
|
5% |
1736.37 |
1157.24 |
|
|
|
10% |
2106.79 |
1517.15 |
1048.67 |
|
|
20% |
2607.09 |
2254.11 |
1735.63 |
1038.87 |
|
30% |
2839.95 |
2649.93 |
2403.10 |
1782.15 |
940.39 |
40% |
|
2763.74 |
2624.11 |
2213.10 |
1279.24 |
50% |
|
|
2738.40 |
2456.48 |
1790.14 |
[0395] As shown in FIG. 45, if the incident angle of incident light was 21.5°, the intensity
of reflected light in area NO. 2 was used for an ethanol concentration range of 0
to 10 per cent; the intensity of reflected light in area NO. 10 was used for an ethanol
concentration range of 10 to 30 per cent; and the intensity of reflected light in
area NO. 19 was used for an ethanol concentration range of 30 to 50 per cent. In this
manner, it was confirmed that assessment could be performed on a wide range of ethanol
concentrations, even if the incident angle of incident light is fixed.
[0396] Likewise, as shown in FIG. 46, if the incident angle of incident light was 21.0°,
the intensity of reflected light in area NO. 10 was used for an ethanol concentration
range of 0 to 10 per cent; the intensity of reflected light in area NO. 18 was used
for an ethanol concentration range of 10 to 30 per cent; and the intensity of reflected
light in area NO. 22 was used for an ethanol concentration range of 30 to 50 per cent.
In this manner, it was confirmed that assessment could be performed on a wide range
of ethanol concentrations, even if the incident angle of incident light is fixed.
[0397] Accordingly, even thought this invention uses an easier measurement technique, where
the incident angle of incident light is fixed and where angle scanning is not performed,
in comparison with a conventional technique, where angle scanning is performed to
measure the intensity of reflected light, it is still possible to perform a wide range
of measurement.
[Example 5]
[0398] On the surface of a flat-shaped substrate of polycarbonate, there were provided diffraction
gratings formed of projections and depressions having a groove pitch of approximately
870 nm and a groove depth of approximately 40 nm.
[0399] As shown in FIG. 47, the substrate was then made into a shape such that a cross section
of the substrate taken along the direction perpendicular to the diffraction grating
made a curved surface having a radius of curvature of 1150 mm and such that the diffraction
grating was placed on the convex side of the substrate. After that, a depth of approximately
80 nm of gold was evaporated onto the surface of the substrate, thus producing a sensor
chip.
[0400] Using the thus produced sensor chip, angle scanning was performed with a resonance
angle detecting type SPR assessment device, FLEX CHIPS™ Kinetic Analysis System (
HTS Biosystems Inc.), to measure the intensity of reflected light. In the measurement, a light beam
of a wavelength of approximately 870 nm was used as incident light, and purified water
served as a sample.
[0401] Table 8 shows detection results of the resonance angle of reflected light detected
at every 0.33 mm-distance point along the direction perpendicular to the diffraction
grating on the surface of the sensor chip.
[Table 8]
|
Curved surface A |
Curved surface B |
|
Resonance angle |
Difference |
Resonance angle |
Difference |
|
20.6269 |
|
20.6369 |
|
20.6302 |
0.0033 |
20.6400 |
0.0032 |
20.6376 |
0.0074 |
20.6481 |
0.0080 |
20.6467 |
0.0091 |
20.6514 |
0.0033 |
20.6520 |
0.0053 |
20.6659 |
0.0145 |
20.6585 |
0.0065 |
20.6695 |
0.0037 |
20.6852 |
0.0267 |
20.6892 |
0.0197 |
20.6887 |
0.0035 |
20.6966 |
0.0074 |
20.6998 |
0.0111 |
20.7020 |
0.0054 |
20.7082 |
0.0085 |
20.7074 |
0.0054 |
20.7204 |
0.0122 |
20.7158 |
0.0084 |
20.7255 |
0.0051 |
20.7262 |
0.0103 |
20.7334 |
0.0078 |
20.7348 |
0.0086 |
20.7562 |
0.0229 |
20.7408 |
0.0060 |
20.7600 |
0.0038 |
20.7626 |
0.0218 |
20.7736 |
0.0135 |
20.7720 |
0.0095 |
20.7878 |
0.0143 |
20.7823 |
0.0103 |
20.8013 |
0.0135 |
20.8006 |
0.0183 |
Average |
|
0.0103 |
|
0.0096 |
[0402] The sum of the difference of angles among planes tangent to those points was 0.17°.
As this value was smaller than the angle that should have been formed by the tangent
planes locating at each of the ends of the diffraction grating area, it is assumed
that the example chip was produced to have a radius of curvature bigger than that
which was originally designed.
[0403] In this example, however, the mean of the angles formed between adjacent areas, each
of which includes one of the above-mentioned 0.33 mm-spaced points, is controlled
to take an extremely small value of 0.010°. It was confirmed that a diffraction grating
surface can be formed on the surface of a sensor chip even if the grating has a curved
surface.
[Example 9]
[0404] On the surface of a flat-shaped substrate of polycarbonate, there were provided diffraction
gratings formed of projections and depressions. A depth of approximately 80 nm of
gold was then evaporated onto the surface of the substrate, thus producing a sensor
chip.
[0405] On this sensor chip, there are provided 2.5 mm-wide areas where diffraction gratings
with separate groove pitches (TP) of, 846 nm, 856 nm, 870 nm, and 876 nm, respectively,
are formed. Using this sensor chip, the intensity of reflected light was measured
with an SPR assessment device, FLEX CHIPS™ Kinetic Analysis System (
HTS Biosystems Inc.), while varying the angle of incident light in a range of 19.89° to 21.18°. Purified
water, 1% ethanol aqueous solution, 10% ethanol aqueous solution, and 30% ethanol
aqueous solution served as samples, which were introduced in order onto the surface
of the sensor chip. In this instance, the temperature of the solutions was 30°C, and
the wavelength of the incident light was 870 nm.
[0406] FIG. 48 is a plot of the measurement result obtained in each of the areas with groove
pitches (TP) of 846 nm, 856 nm, 870 nm, and 876 nm, respectively. The horizontal axis
of the graph represents the time duration elapsed after the measurement was started,
and the vertical axis of the graph represents the resonance angle.
[0407] The part of the graph from 0 through 1200 seconds represents data obtained for purified
water as a sample; that from 1200 through 1400 seconds, 1% ethanol aqueous solution;
that from 1400 through 1720 seconds, 10% ethanol aqueous solution; that from 1720
through 2140 seconds, 30% ethanol aqueous solution; and that at 2140 seconds and afterward,
purified water again.
[0408] From the graph of a groove pitch of 870 nm, it is possible to examine the amount
of variation between the resonance angles for 1% ethanol aqueous solution and 10 %
ethanol aqueous solution and the resonance angle for purified water. However, since
the graph for 30% ethanol aqueous solution extends to the outside of the measurement
range, the amount of difference cannot be measured for 30% ethanol aqueous solution
from the graph of a groove pitch of 870 nm.
[0409] As for the graph of a groove pitch of 856 nm, however, it shows data obtained from
10% ethanol aqueous solution and 30% ethanol aqueous solution. The amount of variation
between the intensity of reflected light for 30% ethanol aqueous solution and that
for 10% ethanol aqueous solution, obtained from the 856 nm groove pitch graph, is
thus used for data correction, thereby obtaining the amount of variation between the
intensity of reflected light for 30% ethanol aqueous solution and that for purified
water. That is, the amount of variation between the intensity of reflected light for
30% ethanol aqueous solution and that for 10% ethanol aqueous solution is added to
the amount of variation between the intensity of reflected light for 10% ethanol aqueous
solution and that for purified water, thereby obtaining the amount of variation between
the intensity of reflected light for 30% ethanol aqueous solution and that for purified
water.
[0410] Table 9 shows the thus-obtained amount of variation between the intensity of reflected
light for ethanol aqueous solutions of such three types of concentrations and that
for purified water.
[Table 9]
Ethanol concentration |
The amount of variation in the resonance
angle in comparison with purified water |
1% |
33 mdeg |
10% |
362 mdeg |
30% |
1143 mdeg |
|
[The amount of variation between the |
|
resonance angle for 10% ethanol aqueous |
|
solution and that for purified water (362 |
|
mdeg) + the amount of variation between the |
|
resonance angle for 30% ethanol aqueous |
|
solution and that 10% EtOH (781 mdeg)] |
[0411] FIG. 49 plots the data of table 9. FIG. 49 indicates linearity between the ethanol
concentration and the amount of variation between the intensity of reflected light
of the sample and that of purified water.
[0412] In this manner, it was confirmed that, by using a sensor chip on which areas where
diffraction gratings with separate groove pitches are formed, it is possible to measure
a wider range of intensities of reflected light with no necessity of greatly varying
the incident angle of incident light, thus realizing assessment with excellent time
resolution.
[Example 10]
[0413] As in the case of Example 1, using a sensor chip on which provided were 2.5-mm-wide
areas where diffraction gratings with separate groove pitches (TP) of, 846 nm, 856
nm, 870 nm, and 876 nm, respectively, are formed, the intensity of reflected light
was measured while irradiating a sample with light at a fixed incident angle. The
measurement was performed with an SPR assessment device, FLEX CHIPS™ Kinetic Analysis
System, and purified water and 10% ethanol aqueous solution were used as samples.
The temperature of the samples was 30°C at the measurement; the wavelength of the
incident light was 875 nm; and the incident angle of the incident light was 20.8502°.
[0414] FIG. 50(a) plots the result of the measurement.
[0415] From this measurement result, groove pitches at which resonance was caused in purified
water and 10% ethanol aqueous solution were obtained by using a 6th-degree polynomial
equation. FIG. 50(b) shows an approximate method in which a polynomial equation is
used, and Table 10 shows its result.
[Table 10]
Ethanol concentration |
Groove pitch |
The amount of variation in the groove pitch in comparison with purified water |
0% (purified water) |
871.72 nm |
0 nm |
10% |
863.30 nm |
8.42 nm |
[0416] Table 10 indicates that the graph of purified water and the graph of 10% ethanol
aqueous solution showed the minimal intensity of reflected light for different groove
pitches. Difference between these groove pitches, or the amount of variation in the
groove pitch, can be used for analyzing samples.
[0417] From the above, it was confirmed that with use of a sensor chip of the present invention,
assessment can be performed with an SPR sensor chip even when the incident angle of
incident light is fixed.
[0418] Although only four types of groove pitch areas were used in this example, a larger
number of types of groove pitches would make more precise assessment possible.
Industrial Applicability
[0419] As described above, surface plasmon resonance sensor chips according to the present
invention are suitable for use in downsized clinical equipment or HPLC (high performance
liquid chromatography) detectors. POC (Point of Care) technology has become increasingly
valued particularly in clinical examination fields because of its compactness and
easy operation so that examinations can be performed in parallel with medical treatment.
The surface plasmon resonance sensor chips of the present invention are also considered
applicable to immunologic tests. Since the surface plasmon resonance sensor chips
of the present invention provide downsized, cost-reduced analysis equipment, they
are applicable to not only POC but also other fields such as housing examination.
Moreover, since the surface plasmon resonance sensor chips of the present invention
are also suitable for use in HPLC, they have wide applicability in the analysis of
a variety of items such as blood or urine, food nutrition, and chemicals contained
in wastewater.
1. A surface plasmon resonance sensor chip comprising:
a metal layer (3) along whose surface a surface plasmon wave can be induced by light
irradiation; and
a plurality of diffraction grating surfaces (5a-5i, 251-254) that are disposed in
the vicinity of said metal layer (3) and on each of which a diffraction grating with
a uniform groove orientation and a uniform groove pitch is formed so as to generate
an evanescent wave upon light irradiation;
wherein the groove pitch (d1-d4) and the groove orientation of each said diffraction
grating surface (5a-5i, 251-254), in addition to the angle (αa-αi) that each said
diffraction grating surface (5a-5i, 251-254) forms with a predetermined reference
plane (S0), are adjusted in such a manner that when said diffraction grating surfaces
(5a-5i, 251-254) are projected onto a predetermined projection plane, the groove orientations
in the projection plane are identical while the groove pitches in the projection plane
are different among said diffraction grating surfaces (5a-5i, 251-254).
2. A surface plasmon resonance sensor chip as defined in claim 1, wherein
each said diffraction grating surface (5a-5i) is disposed so as to be perpendicular
to a specific plane (S1), which is perpendicular to the predetermined reference plane
(S0), and as to form a predetermined inclination angle (αa-αi) with the reference
plane (S0), and
on each said diffraction grating surface (5a-5i), the diffraction grating is formed
in such a manner that the groove orientation is perpendicular to the specific plane
(S1).
3. A surface plasmon resonance sensor chip as defined in claim 2, wherein said plural
diffraction grating surfaces (5a-5i) are disposed along a line parallel to the specific
plane (S1) in such a manner that when viewed from a direction parallel to the specific
plane (S1), said plural diffraction grating surfaces (5a-5i) are positioned in decreasing
order of the inclination angle (αa-αi) that each said diffraction grating surface
(5a-5i) forms with the reference plane (S0).
4. A surface plasmon resonance sensor chip as defined in claim 2 or 3, wherein said diffraction
grating surfaces (5a-5i) are disposed continuously so as to form a convex shape whose
light-irradiated side bulges out.
5. A surface plasmon resonance sensor chip as defined in claim 4, wherein each said diffraction
grating surface (25) has a minimum width with one groove alone, and the aggregate
of said diffraction grating surfaces (25) forms a curved surface in an arc shape whose
light-irradiated side bulges out.
6. A surface plasmon resonance sensor chip as defined in one of claims 2-5, wherein
each said diffraction grating surface (5a-5i) is formed along a sensor surface
(1a), which comes in contact with a sample, and
on the sensor surface (1a), a binding substance (7) that binds specifically to
a target species in the sample is immobilized for each said diffraction grating surface
(5a-5i).
7. A surface plasmon resonance sensor chip as defined in claim 6, wherein two or more
kinds of binding substances (7) are immobilized for each said diffraction grating
surface (5a-5i).
8. A surface plasmon resonance sensor chip as defined in one of claims 2-7, further comprising
a plurality of diffraction areas (6), within each of which said diffraction grating
surfaces (5a-5i) are concentratedly disposed, wherein said plural diffraction grating
surfaces (5a-5i) in each of said diffraction areas (6) have different inclination
angles.
9. A surface plasmon resonance sensor chip as defined in claim 8, wherein
each said diffraction grating surface (5a-5i) is disposed along a sensor surface
(1a), which comes in contact with a sample, and
on the sensor surface (1a), two or more binding substances (7) which each bind
specifically to target species in the sample are immobilized so as to be associated
with said diffraction areas (6).
10. A surface plasmon resonance sensor chip as defined in one of claims 6-9, further comprising
a plurality of non-diffraction surfaces (37a-37i), which do not have any diffraction
grating,
wherein each of said non-diffraction surfaces (37a-37i) is disposed along the sensor
surface in the same plane with the respective one of said diffraction grating surfaces
(35a-35i).
11. A surface plasmon resonance sensor chip as defined in one of claims 6-9, wherein each
said diffraction grating surface has a reaction area, within which the binding substance
(47) is immobilized, and a non-reaction area, within which a substance (48) that does
not bind specifically to any target species in the sample is immobilized or, alternatively,
any substance is not immobilized.
12. A surface plasmon resonance sensor chip as defined in one of claims 6-9, wherein
said diffraction grating surfaces are arranged in a direction perpendicular to
the groove orientation, and
said sensor chip further comprises
a cover (72) for covering the sensor surface (1a), and
a plurality of flow channels (70) formed side by side between the sensor surface
(1a) and said cover (72) so as to pass along the direction in which said diffraction
grating surfaces are arranged.
13. A surface plasmon resonance sensor chip as defined in claim 8 or 9, further comprising
a plurality of non-diffraction areas (88) associated one with each said diffraction
area (87), each of said non-diffraction areas (88) having a plurality of non-diffraction
surfaces concentratedly disposed therein, each of which non-diffraction surfaces does
not have any diffraction grating,
wherein the inclination angles that said non-diffraction surfaces included in the
non-diffraction area (88) form with the reference plane have the same distribution
as the distribution of the inclination angles that said diffraction grating surfaces
included in the associated diffraction area (87) form with the reference plane.
14. A surface plasmon resonance sensor chip as defined in claim 8 or 9, wherein
each of one or more diffraction areas among said diffraction areas (96) has a reaction
area, in which a binding substance (97) that binds specifically to a target species
in the sample is immobilized, and
each of the remaining diffraction areas among said diffraction areas (96) has a
non-reaction area, in which a substance (98) that does not bind specifically to any
target species in the sample is immobilized or, alternatively, any substance is not
immobilized.
15. A surface plasmon resonance sensor chip as defined in claim 8 or 9, further comprising
a cover (102) for covering the sensor surface (1a), and
a plurality of flow channels (100) disposed side by side between the sensor surface
(1a) and said cover (102),
wherein said diffraction areas (6) are disposed for each of said flow channels
(100).
16. A method of quantitatively and/or qualitatively analyzing a sample using a surface
plasmon resonance sensor chip as defined in one of claims 6-9, comprising the steps
of:
making the sample in contact with the sensor surface (1a) while irradiating the sensor
surface (1a) with light in parallel to the specific plane (S1) at a predetermined
incident angle;
receiving the light reflected from the sensor surface (1a) and measuring the intensity
of the light reflected by each said diffraction grating surface (5a-5i);
calculating a resonance angle at which a resonance phenomenon of the evanescent wave
and the surface plasmon wave occurs, based on both the measured intensity of the reflected
light due to each said diffraction grating surface (5a-5i) and the inclination angle
that each said diffraction grating surface (5a-5i) forms with the reference plane
(S0); and
quantitatively and/or qualitatively analyzing the sample based on the calculated resonance
angle.
17. A method of quantitatively and/or qualitatively analyzing a sample using a surface
plasmon resonance sensor chip as defined in claim 10 or 13, comprising the steps of:
making the sample in contact with the sensor surface while irradiating the sensor
surface with light in parallel to the specific plane at a predetermined incident angle;
receiving the light reflected from the sensor surface and measuring the intensity
of the light reflected by each said diffraction grating surface (35a-35i);
correcting the measured intensity of the reflected light due to each said diffraction
grating surface (35a-35i) with consideration given to the intensity of the light reflected
by each said non-diffraction surface (37a-37i);
calculating a resonance angle at which a resonance phenomenon of the evanescent wave
and the surface plasmon wave occurs, based on both the corrected intensity of reflected
light due to each said diffraction grating surface (35a-35i) and the inclination angle
that each said diffraction grating surface (35a-35i) forms with the reference plane;
and
quantitatively and/or qualitatively analyzing the sample based on the calculated resonance
angle.
18. A method of quantitatively and/or qualitatively analyzing a sample using a surface
plasmon resonance sensor chip as defined in claim 11 or 14, comprising the steps of:
making the sample in contact with the sensor surface while irradiating the sensor
surface with light in parallel to the specific plane at a predetermined incident angle;
receiving the light reflected from the sensor surface and measuring the intensity
of the light reflected by each said diffraction grating surface;
calculating, for each of the reaction area and the non-reaction area, a resonance
angle at which a resonance phenomenon of the evanescent wave and the surface plasmon
wave occurs, based on both the measured intensity of the reflected light due to each
said diffraction grating surface and the inclination angle that each said diffraction
grating surface forms with the reference plane; and
after correcting the resonance angle of the reaction area with consideration given
to the resonance angle of the non-reaction area, quantitatively and/or qualitatively
analyzing the sample based on the corrected resonance angle of the reaction area.
19. A method of quantitatively and/or qualitatively analyzing a sample using a surface
plasmon resonance sensor chip as defined in claim 12 or 15, comprising the steps of:
assigning a plurality of different samples to said plural flow channels (70), respectively,
and letting each of the samples flow through the respective flow channel (70) while
irradiating the sensor surface with light in parallel to the specific plane at a predetermined
incident angle;
receiving the light reflected from the sensor surface and measuring the intensity
of the light reflected by each said diffraction grating surface;
calculating, for each sample flowing through the respective flow channel (70), a resonance
angle at which a resonance phenomenon of the evanescent wave and the surface plasmon
wave occurs, based on both the measured intensity of the reflected light due to each
said diffraction grating surface and the inclination angle that each said diffraction
grating surface forms with the reference plane; and
quantitatively and/or qualitatively analyzing each sample flowing through the respective
flow channel (70), based on the calculated resonance angle for each said flow channel
(70).
20. A method of quantitatively and/or qualitatively analyzing a sample using a surface
plasmon resonance sensor chip as defined in one of claim 6-9, comprising the steps
of:
making the sample in contact with the sensor surface (1a) while irradiating the sensor
surface (1a) with light in parallel to the specific plane (S1) at a predetermined
incident angle;
receiving the light reflected from the sensor surface (1a) and measuring the intensity
of the light reflected by each said diffraction grating surface (5a-5i);
determining the variation between the measured intensity of the reflected light due
to each said diffraction grating surface (5a-5i) and the intensity of the light reflected
when any sample is not in contact with the sensor surface (S1); and
selecting a diffraction grating surface (5a-5i) whose determined variation of the
reflected-light intensity is within a predetermined allowable range for determination,
and quantitatively and/or qualitatively analyzing the sample based on the variation
of the reflected-light intensity of the selected diffraction grating surface (5a-5i).
21. A method of quantitatively and/or qualitatively analyzing a sample using a surface
plasmon resonance sensor chip as defined in claim 10 or 13, comprising the steps of:
making the sample in contact with the sensor surface while irradiating the sensor
surface with light in parallel to the specific plane at a predetermined incident angle;
receiving the light reflected from the sensor surface and measuring the intensity
of the light reflected by each said diffraction grating surface (35a-35i);
correcting the intensity of the reflected light due to each said diffraction grating
surface (35a-35i) with consideration given to the intensity of the reflected light
due to each said non-diffraction surface (37a-37i);
determining the variation between the corrected intensity of the reflected light due
to each said diffraction grating surface (35a-35i) and the intensity of the light
reflected when any sample is not in contact with the sensor surface; and
selecting a diffraction grating surface (35a-35i) whose determined variation of the
reflected-light intensity is within a predetermined allowable range for determination,
and quantitatively and/or qualitatively analyzing the sample based on the variation
of the reflected-light'intensity of the selected diffraction grating surface (35a-35i).
22. A method of quantitatively and/or qualitatively analyzing a sample using a surface
plasmon resonance sensor chip as defined in claim 11 or 14, comprising the steps of:
making the sample in contact with the sensor surface while irradiating the sensor
surface with light in parallel to the specific plane at a predetermined incident angle;
receiving the light reflected from the sensor surface and measuring the intensity
of the light reflected by each said diffraction grating surface;
determining, for each of the reaction area and the non-reaction area, the variation
between the measured intensity of the reflected light due to each said diffraction
grating surface and the intensity of the light reflected when any sample is not in
contact with the sensor surface; and
selecting, for each of the reaction area and the non-reaction area, a diffraction
grating surface whose determined variation of the reflected-light intensity is within
a predetermined allowable range for determination, and quantitatively and/or qualitatively
analyzing the sample based on both the selected variation of the reflected-light intensity
of the reaction area and the selected variation of the reflected-light intensity of
the non-reaction area.
23. A method of quantitatively and/or qualitatively analyzing a sample using a surface
plasmon resonance sensor chip as defined in claim 12 or 15, comprising the steps of:
assigning a plurality of different samples to said plural flow channels (70), respectively,
and letting each of the samples flow through the respective flow channel (70) while
irradiating the sensor surface with light in parallel to the specific plane at a predetermined
incident angle;
receiving the light reflected from the sensor surface and measuring the intensity
of the light reflected by each said diffraction grating surface;
determining the variation between the measured intensity of the reflected light due
to each said diffraction grating surface and the intensity of the light reflected
when any sample does not flow through each said flow channel (70); and
selecting, for each said flow channel (70), a diffraction grating surface whose determined
variation of the reflected-light intensity is within a predetermined allowable range
for determination, and quantitatively and/or qualitatively analyzing each sample flowing
through the respective flow channel (70), based on the variation of the reflected-light
intensity of the diffraction grating surface selected for each said flow channel (70).
24. A method as defined in one of claims 16-23, further comprising the step of separating
the sample by physical and/or chemical action prior to introducing the sample to the
surface plasmon resonance sensor chip.
25. A method as defined in claim 24, wherein in said step of separating, the sample is
separated by a separation technique using at least one of liquid chromatography, HPLC
(high performance liquid chromatography), capillary electrophoresis, microchip electrophoresis,
flow injection, and microchannel.
26. A method as defined in one of claim 16-25, wherein
the target species is a light-emitting substance,
said method further comprises the step of detecting the light emitted from the
light-emitting substance that is bound to the binding substance prior to light-irradiating
the sensor surface or, alternatively, after light-irradiating the sensor surface and
receiving the reflected light, and
in said step of quantitatively and/or qualitatively analyzing, the sample is analyzed
with consideration given to the detection result of the emitted light.
27. An apparatus for quantitatively and/or qualitatively analyzing a sample using a surface
plasmon resonance sensor chip as defined in one of claims 6-9, comprising:
holding means (11) for holding the surface plasmon resonance sensor chip with the
sensor surface being in contact with the sample;
light irradiating means (12) for irradiating the sensor surface (1a) with light in
parallel to the specific plane (S1) at a predetermined incident angle in a state where
the surface plasmon resonance sensor chip is held by said holding means (11);
light receiving means (13) for receiving the light reflected from the sensor surface
(1a);
measuring means (13) for measuring the intensity of the light reflected by each said
diffraction grating surface (1a) and received by said light receiving means (13);
calculating means (14) for calculating a resonance angle at which a resonance phenomenon
of the evanescent wave and the surface plasmon wave occurs, based on both the intensity,
measured by said measuring means (13), of the reflected light due to each said diffraction
grating surface (1a) and the inclination angle that each said diffraction grating
surface (1a) forms with the reference plane (S0); and
analyzing means for quantitatively and/or qualitatively analyzing the sample based
on the resonance angle calculated by said calculating means (14).
28. An apparatus for quantitatively and/or qualitatively analyzing a sample using a surface
plasmon resonance sensor chip as defined in claim 10 or 13, comprising:
holding means (11) for holding the surface plasmon resonance sensor chip with the
sensor surface being in contact with the sample;
light irradiating means (12) for irradiating the sensor surface with light in parallel
to the specific plane at a predetermined incident angle in a state where the surface
plasmon resonance sensor chip is held by said holding means (11);
light receiving means (13) for receiving the light reflected from the sensor surface;
measuring means (13) for measuring the intensity of the light reflected by each said
diffraction grating surface and received by said light receiving means (13);
correcting means (14) for correcting the intensity of reflected light due to each
said diffraction grating surface (35a-35i) with consideration given to the intensity
of the reflected light due to the non-diffraction surface (37a-37i);
calculating means (14) for calculating a resonance angle at which a resonance phenomenon
of the evanescent wave and the surface plasmon wave occurs, based on both the intensity,
corrected by said correcting means (14), of the reflected light due to each said diffraction
grating surface (35a-35i) and the inclination angle that each said diffraction grating
surface (35a-35i) forms with the reference plane; and
analyzing means for quantitatively and/or qualitatively analyzing the sample based
on the resonance angle calculated by said calculating means (14).
29. An apparatus for quantitatively and/or qualitatively analyzing a sample using a surface
plasmon resonance sensor chip as defined in claim 11 or 14, comprising:
holding means (11) for holding the surface plasmon resonance sensor chip with the
sensor surface being in contact with the sample;
light irradiating means (12) for irradiating the sensor surface with light in parallel
to the specific plane at a predetermined incident angle in a state where the surface
plasmon resonance sensor chip is held by said holding means (11);
light receiving means (13) for receiving the light reflected from the sensor surface;
measuring means (13) for measuring the intensity of the light reflected by each said
diffraction grating surface and received by said light receiving means (13);
calculating means (14) for calculating, for each of the reaction area and the non-reaction
area, a resonance angle at which a resonance phenomenon of the evanescent wave and
the surface plasmon wave occurs, based on the intensity, measured by said measuring
means (13), of the reflected light due to each said diffraction grating surface and
the inclination angle that each said diffraction grating surface forms with the reference
plane; and
analyzing means (14) for correcting the resonance angle, calculated by said calculating
means (14), of the reaction area with consideration given to the resonance angle of
the non-reaction area and for quantitatively and/or qualitatively analyzing the sample
based on the corrected resonance angle of the reaction area,.
30. An apparatus for quantitatively and/or qualitatively analyzing a sample using a surface
plasmon resonance sensor chip as defined in claim 12 or 15, comprising:
holding means (11) for holding the surface plasmon resonance sensor chip;
sample introducing means (79) for assigning a plurality of different samples to said
plural flow channels (70), respectively, and for introducing each of the samples into
the respective flow channel (70) in a state where the surface plasmon resonance sensor
chip is held by said holding means (11);
light irradiating means (12) for irradiating the sensor surface with light in parallel
to the specific plane at a predetermined incident angle in a state where each sample
is introduced into the respective flow channel (70) by said sample introducing means
(79);
light receiving means (13) for receiving the light reflected from the sensor surface;
measuring means (13) for measuring the intensity of the light reflected by each said
diffraction grating surface and received by said light receiving means (13);
calculating means (14) for calculating a resonance angle at which a resonance phenomenon
of the evanescent wave and the surface plasmon wave occurs for each said flow channel
(70), based on the intensity, measured by said measuring means (13), of the reflected
light due to each said diffraction grating surface and the inclination angle that
each said diffraction grating surface forms with the reference plane; and
analyzing means (14) for quantitatively and/or qualitatively analyzing each sample
flowing through the respective flow channel (70), based on the resonance angle calculated
by said calculating means (14).
31. An apparatus for quantitatively and/or qualitatively analyzing a sample using a surface
plasmon resonance sensor chip as defined in one of claims 6-9, comprising:
holding means (11) for holding the surface plasmon resonance sensor chip with the
sensor surface being in contact with the sample;
light irradiating means (12) for irradiating the sensor surface with light in parallel
to the specific plane at a predetermined incident angle in a state where the surface
plasmon resonance sensor chip is held by said holding means (11);
light receiving means (13) for receiving the light reflected from the sensor surface;
measuring means (13) for measuring the intensity of the light reflected by each said
diffraction grating surface (5a-5i) and received by said light receiving means (13);
determining means (14) for determining the variation between the intensity, measured
by said measuring means (13), of the reflected light due to each said diffraction
grating surface (5a-5i) and the intensity of the light reflected when any sample is
not in contact with the sensor surface (1a);
analyzing means for selecting a diffraction grating surface (5a-5i) whose variation,
determined by said determining means (14), of the reflected-light intensity is within
a predetermined allowable range for determination, and for quantitatively and/or qualitatively
analyzing the sample based on the variation of the reflected-light intensity of the
selected diffraction grating surface (5a-5i).
32. An apparatus for quantitatively and/or qualitatively analyzing a sample using a surface
plasmon resonance sensor chip as defined in claim 10 or 13, comprising:
holding means (11) for holding the surface plasmon resonance sensor chip with the
sensor surface being in contact with the sample;
light irradiating means (12) for irradiating the sensor surface with light in parallel
to the specific plane at a predetermined incident angle in a state where the surface
plasmon resonance sensor chip is held by said holding means (11);
light receiving means (13) for receiving the light reflected from the sensor surface;
measuring means (13) for measuring the intensity of the light reflected by each said
diffraction grating surface and received by said light receiving means (13);
correcting means (14) for correcting the intensity of reflected light due to each
said diffraction grating surface (35a-35i) with consideration given to the intensity
of the reflected light due to the non-diffraction surface (37a-37i);
determining means (14) for determining the variation between the intensity, corrected
by said correcting means (14), of the reflected light due to each said diffraction
grating surface (35a-35i) and the intensity of the light reflected when any sample
is not in contact with the sensor surface; and
analyzing means (14) for selecting a diffraction grating surface (35a-35i) whose variation,
determined by said determining means (14), of the reflected-light intensity is within
a predetermined allowable range for determination, and for quantitatively and/or qualitatively
analyzing the sample based on the variation of the reflected-light intensity of the
selected diffraction grating surface (35a-35i).
33. An apparatus for quantitatively and/or qualitatively analyzing a sample using a surface
plasmon resonance sensor chip as defined in claim 11 or 14, comprising:
holding means (11) for holding the surface plasmon resonance sensor chip with the
sensor surface being in contact with the sample;
light irradiating means (12) for irradiating the sensor surface with light in parallel
to the specific plane at a predetermined incident angle in a state where the surface
plasmon resonance sensor chip is held by said holding means (11);
light receiving means (13) for receiving the light reflected from the sensor surface;
measuring means (13) for measuring the intensity of the light reflected by each said
diffraction grating surface and received by said light receiving means (13);
determining means (14) for determining, for each of the reaction area and the non-reaction
area, the variation between the intensity, measured by said measuring means (13),
of the reflected light due to each said diffraction grating surface and the intensity
of the light reflected when any sample is not in contact with the sensor surface;
and
analyzing means (14) for selecting a diffraction grating surface whose variation,
determined by said determining means (14), of the reflected-light intensity is within
a predetermined allowable range for determination, and for quantitatively and/or qualitatively
analyzing the sample based on both the variation of the reflected-light intensity
of the selected reaction area and the variation of the reflected-light intensity of
the selected non-reaction area.
34. An apparatus for quantitatively and/or qualitatively analyzing a sample using a surface
plasmon resonance sensor chip as defined in claim 12 or 15, comprising:
holding means (11) for holding the surface plasmon resonance sensor chip;
sample introducing means (79) for assigning a plurality of different samples to the
plural flow channels (70), respectively, and for introducing each of the samples into
the respective flow channel (70) in a state where the surface plasmon resonance sensor
chip is held by said holding means (11);
light irradiating means (12) for irradiating the sensor surface with light in a predetermined
direction in a state where each sample is introduced into the respective flow channel
(70) by said sample introducing means (79);
light receiving means (13) for receiving the light reflected from the sensor surface;
determining means (14) for determining the variation between the intensity of the
light reflected by each said diffraction grating surface and received by said light
receiving means (13) and the intensity of the light reflected when any sample is not
flowing through each said flow channel (70); and
analyzing means (14) for selecting, for each said flow channel (70), a diffraction
grating surface whose variation, determined by said determining means (14), of the
reflected-light intensity is within a predetermined allowable range for determination,
and for quantitatively and/or qualitatively analyzing each sample flowing through
the respective flow channel (70), based on the variation of the reflected-light intensity
of the diffraction grating surface selected for each said flow channel (70).
35. An apparatus as defined in one of claim 27-34, further comprising sample separating
means (59) for separating the sample by physical and/or chemical action prior to introducing
the sample to the surface plasmon resonance sensor chip.
36. An apparatus as defined in claim 35, wherein said sample separating means (59) is
operable to separate the sample by a separation technique using at least one of liquid
chromatography, HPLC (high performance liquid chromatography), capillary electrophoresis,
microchip electrophoresis, flow injection, and microchannel.
37. An apparatus as defined in one of claim 27-36, wherein
the target species is a light-emitting substance,
said light receiving means (13) is operable to detect the light emitted from the
light-emitting substance that is bound to the binding substance, and
said analyzing means (14) is operable to quantitatively and/or qualitatively analyze
the sample with consideration given to the detection result of the light emission
by said light receiving means (13).
38. A surface plasmon resonance sensor chip as defined in claim 1, wherein
each said diffraction grating surface (251-254) is disposed so as to be parallel
to a predetermined reference plane, and
on each said diffraction grating surface (251-254), the diffraction grating (205)
is formed in such a manner that the groove orientations are identical while the groove
pitches (d1-d4) are different among said diffraction grating surfaces (251-254).
39. A surface plasmon resonance sensor chip as defined in claim 38, wherein
each said diffraction grating surface (251-254) is formed along a sensor surface
(201a), which comes in contact with a sample, and
on the sensor surface (201a), a binding substance (206) that binds specifically
to a target species in the sample is immobilized for each said diffraction grating
surface (251-254).
40. A surface plasmon resonance sensor chip as defined in claim 39, wherein two or more
kinds of binding substances (206, 207) are immobilized for each said diffraction grating
surface (251-254).
41. A surface plasmon resonance sensor chip as defined in claim 49 or 40, further comprising
a plurality of non-diffraction surfaces (251x-254x), each of which does not have any
diffraction grating,
wherein each of said non-diffraction surfaces (251x-254x) is disposed along the
sensor surface (261a) in the same plane with the respective one of said diffraction
grating surfaces (251-254).
42. A surface plasmon resonance sensor chip as defined in claim 39 or 40, wherein each
said diffraction grating surface (251-254) has a reaction area, within which the binding
substance (206) is immobilized, and a non-reaction area, within which a substance
(291) that does not bind specifically to any target species in the sample is immobilized
or, alternatively, any substance is not immobilized.
43. A surface plasmon resonance sensor chip as defined in claim 39 or 40, wherein
said diffraction grating surfaces (251-254) are arranged in a direction perpendicular
to the groove orientation, and
said sensor chip further comprises
a cover (286) for covering the sensor surface (281a), and
a plurality of flow channels (280) formed side by side between the sensor surface
(281a) and said cover (286) so as to pass along the direction in which said diffraction
grating surfaces (281a) are arranged.
44. A surface plasmon resonance sensor chip as defined in claim 39 or 40, wherein:
said diffraction grating surfaces (251-254) are arranged in a direction perpendicular
to the groove orientation;
said sensor chip further comprises
a cover (286) for covering the sensor surface (281a), and
a plurality of flow channels (280) formed side by side between the sensor surface
(281a) and said cover (286) so as to pass along the direction in which said diffraction
grating surfaces (251-254) are arranged; and
along each of said flow channels (280), each said diffraction grating surface (251-254)
has a reaction area, within which the binding substance (206) is immobilized, and
a non-reaction area, within which a substance (291) that does not bind specifically
to any target species in the sample is immobilized or, alternatively, any substance
is not immobilized.
45. A method of quantitatively and/or qualitatively analyzing a sample using a surface
plasmon resonance sensor chip as defined in claim 39 or 40, comprising the steps of:
making the sample in contact with the sensor surface (201a) while irradiating the
sensor surface (201a) with light at a predetermined incident angle;
receiving the light reflected from the sensor surface (201a) and measuring the intensity
of the light reflected by each said diffraction grating surface;
identifying a groove pitch at which a resonance phenomenon of the evanescent wave
and the surface plasmon wave occurs, based on the measured intensity of the reflected
light due to each said diffraction grating surface (201a); and
quantitatively and/or qualitatively analyzing the sample based on the identified groove
pitch.
46. A method of quantitatively and/or qualitatively analyzing a sample using a surface
plasmon resonance sensor chip as defined in claim 41, comprising the steps of:
making the sample in contact with the sensor surface while irradiating the sensor
surface with light at a predetermined incident angle;
receiving the light reflected from the sensor surface and measuring the intensity
of the light reflected by each said diffraction grating surface (251-254);
correcting the intensity of the reflected light due to each said diffraction grating
surface (251-254) with consideration given to the intensity of the light reflected
by the respective non-diffraction surface (251x-254x);
identifying a groove pitch at which a resonance phenomenon of the evanescent wave
and the surface plasmon wave occurs, based on the corrected intensity of the reflected
light due to each said diffraction grating surface (251-254); and
quantitatively and/or qualitatively analyzing the sample based on the identified groove
pitch.
47. A method of quantitatively and/or qualitatively analyzing a sample using a surface
plasmon resonance sensor chip as defined in claim 42, comprising the steps of:
making the sample in contact with the sensor surface while irradiating the sensor
surface with light at a predetermined incident angle;
receiving the light reflected from the sensor surface and measuring the intensity
of the light reflected by each said diffraction grating surface (251-254);
identifying, for each of the reaction area and the non-reaction area, a groove pitch
at which a resonance phenomenon of the evanescent wave and the surface plasmon wave
occurs, based on the measured intensity of the reflected light due to each said diffraction
grating surface (251-254); and
quantitatively and/or qualitatively analyzing the sample based on the groove pitch
identified for each of the reaction area and the non-reaction area.
48. A method of quantitatively and/or qualitatively analyzing a sample using a surface
plasmon resonance sensor chip as defined in claim 43, comprising the steps of:
assigning a plurality of different samples to said plural flow channels (280), respectively,
and letting each of the samples flow through the respective flow channel (280) while
irradiating the sensor surface with light at a predetermined incident angle;
receiving the light reflected from the sensor surface and measuring the intensity
of the light reflected by each said diffraction grating surface (251-254);
identifying, for each said flow channel (280), a groove pitch at which the resonance
phenomenon of the evanescent wave and the surface plasmon wave occurs, based on the
measured intensity of the reflected light due to each said diffraction grating surface
(251-254); and
quantitatively and/or qualitatively analyzing each sample flowing through the respective
flow channel (280), based on the groove pitch identified for each said flow channel
(280).
49. A method of quantitatively and/or qualitatively analyzing a sample using a surface
plasmon resonance sensor chip as defined in claim 44, comprising the steps of:
assigning a plurality of different samples to said plural flow channels (280), respectively,
and letting each of the samples flow through the respective flow channel (280) while
irradiating the sensor surface with light at a predetermined incident angle;
receiving the light reflected from the sensor surface and measuring the intensity
of the light reflected by each said diffraction grating surface (251-254);
identifying, for each said flow channel (280) and for each of the reaction area and
the non-reaction area, a groove pitch at which the resonance phenomenon of the evanescent
wave and the surface plasmon wave occurs, based on the measured intensity of the reflected
light due to each said diffraction grating surface (251-254); and
quantitatively and/or qualitatively analyzing each sample flowing through the respective
flow channel (280), based on the groove pitch identified for each said flow channel
(280) and for each of the reaction area and the non-reaction area.
50. A method of quantitatively and/or qualitatively analyzing a sample using a surface
plasmon resonance sensor chip as define in claim 39 or 40, comprising the steps of:
making the sample in contact with the sensor surface (201a) while irradiating the
sensor surface (201a) with light at a predetermined incident angle;
receiving the light reflected from the sensor surface (201a) and measuring the intensity
of the light reflected by each said diffraction grating surface (251-254);
determining the variation between the measured intensity of the reflected light due
to each said diffraction grating surface (251-254) and the intensity of the light
reflected when any sample is not in contact with the sensor surface (201a); and
selecting a diffraction grating surface (251-254) whose determined variation of the
reflected-light intensity is within a predetermined allowable range for determination,
and quantitatively and/or qualitatively analyzing the sample based on the variation
of the reflected-light intensity of the selected diffraction grating surface (251-254).
51. A method of quantitatively and/or qualitatively analyzing a sample using a surface
plasmon resonance sensor chip as defined in claim 41, comprising the steps of:
making the sample in contact with the sensor surface while irradiating the sensor
surface with light at a predetermined incident angle;
receiving the light reflected from the sensor surface and measuring the intensity
of the light reflected by each said diffraction grating surface (251-254);
correcting the intensity of the reflected light due to each said diffraction grating
surface (251-254) with consideration given to the intensity of the light reflected
by the respective non-diffraction surface (251x-254x);
determining the variation between the corrected intensity of the reflected light due
to each said diffraction grating surface (251-254) and the intensity of the light
reflected when any sample is not in contact with the sensor surface; and
selecting a diffraction grating surface (251-254) whose determined variation of the
reflected-light intensity is within a predetermined allowable range for determination,
and quantitatively and/or qualitatively analyzing the sample based on the variation
of the reflected-light intensity of the selected diffraction grating surface.
52. A method of quantitatively and/or qualitatively analyzing a sample using a surface
plasmon resonance sensor chip as defined in claim 42, comprising the steps of:
making the sample in contact with the sensor surface while irradiating the sensor
surface with light at a predetermined incident angle;
receiving the light reflected from the sensor surface and measuring the intensity
of the light reflected by each said diffraction grating surface (251-254);
determining, for each of the reaction area and the non-reaction area, the variation
between the measured intensity of the reflected light due to each said diffraction
grating surface (251-254) and the intensity of the light reflected when any sample
is not in contact with the sensor surface;
selecting, for each of the reaction area and the non-reaction area, a diffraction
grating surface (251-254) whose determined variation of the reflected-light intensity
is within a predetermined allowable range for determination, and quantitatively and/or
qualitatively analyzing the sample based on the variation of the reflected-light intensity
of the selected reaction area and the variation of the reflected-light intensity of
the selected non-reaction area.
53. A method of quantitatively and/or qualitatively analyzing a sample using a surface
plasmon resonance sensor chip as defined in claim 43, comprising the steps of:
assigning a plurality of different samples to said plural flow channels (280), respectively,
and letting each of the samples flow through the respective flow channel (280) while
irradiating the sensor surface with light at a predetermined incident angle;
receiving the light reflected from the sensor surface and measuring the intensity
of the light reflected by each said diffraction grating surface (251-254);
determining the variation between the measured intensity of the reflected light due
to each said diffraction grating surface (251-254) and the intensity of the light
reflected when any sample does not flow through each said flow channel (280);
selecting, for each said flow channels (280), a diffraction grating surface (251-254)
whose determined variation of the reflected-light intensity is within a predetermined
allowable range for determination, and quantitatively and/or qualitatively analyzing
each sample flowing through the respective flow channel (280), based on the variation
of the reflected-light intensity of the selected diffraction grating surface (251-254)
for each said flow channels (280).
54. A method of quantitatively and/or qualitatively analyzing a sample using a surface
plasmon resonance sensor chip as defined in claim 44, comprising the steps of:
assigning a plurality of different samples to said plural flow channels (280), respectively,
and letting each of the samples flow through the respective flow channel (280) while
irradiating the sensor surface with light at a predetermined incident angle;
receiving the light reflected from the sensor surface and measuring the intensity
of the light reflected by each said diffraction grating surface (251-254);
determining, for each of the reaction area and the non-reaction area, the variation
between the measured intensity of the reflected light due to each said diffraction
grating surface (251-254) and the intensity of the light reflected when any sample
does not flow through each said flow channel (280);
selecting, for each of the flow channels (280) and for each of the reaction area and
the non-reaction area, a diffraction grating surface (251-254) whose determined variation
of the reflected-light intensity is within a predetermined allowable range for determination,
and quantitatively and/or qualitatively analyzing each sample flowing through the
respective flow channel (280), based on the variation of the reflected-light intensity
of the selected reaction area and the variation of the reflected-light intensity of
the selected non-reaction area.
55. A method as defined in one of claims 45-54, wherein it further comprises the step
of separating the sample by physical and/or chemical action prior to introducing the
sample to the surface plasmon resonance sensor chip.
56. A method as defined in claim 55, wherein in said step of separating, the sample is
separated by a separation technique using at least one of liquid chromatography, HPLC
(high performance liquid chromatography), capillary electrophoresis, microchip electrophoresis,
flow injection, and microchannel.
57. A method as defined in one of claims 45-56, wherein
the target species is a light-emitting substance,
said method further comprises the step of detecting the light emitted from the
light-emitting substance that is bound to the binding substance prior to light-irradiating
the sensor surface or, alternatively, after light-irradiating the sensor surface and
receiving the reflected light, and
in said step of quantitatively and/or qualitatively analyzing, the sample is analyzed
with consideration given to the detection result of the light emission.
58. An apparatus for quantitatively and/or qualitatively analyzing a sample using a surface
plasmon resonance sensor chip as defined in claim 39 or 40, comprising:
holding means (211) for holding the surface plasmon resonance sensor chip with the
sensor surface (201a) being in contact with the sample;
light irradiating means (212) for irradiating the sensor surface (201a) with light
from a predetermined direction in a state where the surface plasmon resonance sensor
chip is held by said holding means (211);
light receiving means (213) for receiving the light reflected from the sensor surface;
measuring means (213) for measuring the intensity of the light reflected by each said
diffraction grating surface (251-254) and received by said light receiving means (213);
and
analyzing means (214) for identifying a groove pitch at which a resonance phenomenon
of the evanescent wave and the surface plasmon wave occurs, based on the intensity,
measured by said measuring means (213), of the reflected light due to each said diffraction
grating surface (251-254), and for quantitatively and/or qualitatively analyzing the
sample based on the identified groove pitch.
59. An apparatus for quantitatively and/or qualitatively analyzing a sample using a surface
plasmon resonance sensor chip as define in claim 41, comprising:
holding means (211) for holding the surface plasmon resonance sensor chip with the
sensor surface being in contact with the sample;
light irradiating means (212) for irradiating the sensor surface with light from a
predetermined direction in a state where the surface plasmon resonance sensor chip
is held by said holding means (211);
light receiving means (213) for receiving the light reflected from the sensor surface;
measuring means (213) for measuring the intensity of the light reflected by each said
diffraction grating surface (251-254) and received by said light receiving means (213);
and
correcting means (214) for correcting the intensity of the reflected light due to
each said diffraction grating surface (251-254) with consideration given to the intensity
of the reflected light due to the respective non-diffraction surface (251x-254x);
and
analyzing means (214) for identifying a groove pitch at which a resonance phenomenon
of the evanescent wave and the surface plasmon wave occurs, based on the intensity,
corrected by said correcting means (214), of the reflected light due to each said
diffraction grating surface (251-254), and for quantitatively and/or qualitatively
analyzing the sample based on the identified groove pitch.
60. An apparatus for quantitatively and/or qualitatively analyzing a sample using a surface
plasmon resonance sensor chip as defined in claim 42, comprising:
holding means (211) for holding the surface plasmon resonance sensor chip with the
sensor surface being in contact with the sample;
light irradiating means (212) for irradiating the sensor surface with light from a
predetermined direction in a state where the surface plasmon resonance sensor chip
is held by said holding means (211);
light receiving means (213) for receiving the light reflected from the sensor surface;
measuring means (213) for measuring the intensity of the light reflected by each said
diffraction grating surface (251-254) and received by said light receiving means (213);
and
analyzing means (214) for identifying, for each of the reaction area and the non-reaction
area, a groove pitch at which a resonance phenomenon of the evanescent wave and the
surface plasmon wave occurs, based on the intensity, measured by said measuring means
(213), of the reflected light due to each said diffraction grating surface (251-254),
and for quantitatively and/or qualitatively analyzing the sample based on the groove
pitch identified for each of the reaction area and the non-reaction area.
61. An apparatus for quantitatively and/or qualitatively analyzing a sample using a surface
plasmon resonance sensor chip as defined in claim 43, comprising:
holding means (211) for holding the surface plasmon resonance sensor chip;
sample introducing means (282) for assigning a plurality of different samples to said
plural flow channels (280), respectively, and for introducing each of the plural samples
into the respective flow channel (280) in a state where the surface plasmon resonance
sensor chip is held by said holding means (211);
light irradiating means (212) for irradiating the sensor surface with light from a
predetermined direction in a state where each sample is introduced into the respective
flow channel (282) by said sample introducing means (282);
light receiving means (213) for receiving the light reflected from the sensor surface;
measuring means (213) for measuring the intensity of the light reflected by each said
diffraction grating surface (251-254) and received by said light receiving means (213);
analyzing means for identifying, for each said flow channel (280), a groove pitch
at which a resonance phenomenon of the evanescent wave and the surface plasmon wave
occurs, based on the intensity, measured by said measuring means (213), of the reflected
light due to each said diffraction grating surface (251-254), and for quantitatively
and/or qualitatively analyzing each sample flowing through the respective flow channel
(280), based on the groove pitch identified for each said flow channel (280).
62. An apparatus for quantitatively and/or qualitatively analyzing a sample using a surface
plasmon resonance sensor chip as defined in claim 44, comprising:
holding means (211) for holding the surface plasmon resonance sensor chip;
sample introducing means (282) for assigning a plurality of different samples to said
plural flow channels (280), respectively, and for introducing each sample into the
respective flow channel (280) in a state where the surface plasmon resonance sensor
chip is held by said holding means (211);
light irradiating means (212) for irradiating the sensor surface with light from a
predetermined direction in a state where each sample is introduced into the respective
flow channel (280) by said sample introducing means (282);
light receiving means (213) for receiving the light reflected from the sensor surface;
measuring means (213) for measuring the intensity of the light reflected by each said
diffraction grating surface (251-254) and received by said light receiving means (213);
analyzing means for identifying, for each said flow channel (280) and for each of
the reaction area and the non-reaction area, a groove pitch at which a resonance phenomenon
of the evanescent wave and the surface plasmon wave occurs, based on the intensity,
measured by said measuring means (213), of the reflected light due to each said diffraction
grating surface (251-254), and for quantitatively and/or qualitatively analyzing each
sample flowing through the respective flow channel (280), based on the groove pitches
of the reaction area and the non-reaction area identified for each said flow channel
(280).
63. An apparatus for quantitatively and/or qualitatively analyzing a sample using a surface
plasmon resonance sensor chip as defined in claim 39 or 40, comprising:
holding means (211) for holding the surface plasmon resonance sensor chip with the
sensor surface (201a) being in contact with the sample;
light irradiating means (212) for irradiating the sensor surface with light from a
predetermined direction in a state where the surface plasmon resonance sensor chip
is held by said holding means (211) ;
light receiving means (213) for receiving the light reflected from the sensor surface;
measuring means (213) for measuring the intensity of the light reflected by each said
diffraction grating surface (251-254) and received by said light receiving means (213);
determining means (214) for determining the variation between the intensity, measured
by said measuring means (213), of the reflected light due to each said diffraction
grating surface (251-254) and the intensity of the light reflected when any sample
is not in contact with the sensor surface (201a); and
analyzing means (214) for selecting a diffraction grating surface (251-254) whose
variation, determined by said determining means (214), of the reflected-light intensity
is within a predetermined allowable range for determination, and for quantitatively
and/or qualitatively analyzing the sample based on the variation of the reflected-light
intensity of the selected diffraction grating surface (251-254).
64. An apparatus for quantitatively and/or qualitatively analyzing a sample using a surface
plasmon resonance sensor chip as defined in claim 41, comprising:
holding means (211) for holding the surface plasmon resonance sensor chip with the
sensor surface being in contact with the sample;
light irradiating means (212) for irradiating the sensor surface with light from a
predetermined direction in a state where the surface plasmon resonance sensor chip
is held by said holding means (211);
light receiving means (213) for receiving the light reflected from the sensor surface;
measuring means (213) for measuring the intensity of the light reflected by each said
diffraction grating surface (251-254) and received by said light receiving means (213);
correcting means (214) for correcting the intensity of the reflected light due to
each said diffraction grating surface (251-254) with consideration given to the intensity
of the reflected light due to the respective non-diffraction surface (251x-254x);
determining means (214) for determining the variation between the intensity, corrected
by said correcting means (214), of the reflected light due to each said diffraction
grating surface (251-254) and the intensity of the light reflected when any sample
is not in contact with the sensor surface;
analyzing means (214) for selecting a diffraction grating surface (251-254) whose
variation, determined by said determining means (214), of the reflected-light intensity
is within a predetermined allowable range for determination, and for quantitatively
and/or qualitatively analyzing the sample based on the variation of the reflected-light
intensity of the selected diffraction grating surface (251-254).
65. An apparatus for quantitatively and/or qualitatively analyzing a sample using a surface
plasmon resonance sensor chip as defined in claim 42, comprising:
holding means (211) for holding the surface plasmon resonance sensor chip with the
sensor surface being in contact with the sample;
light irradiating means (212) for irradiating the sensor surface with light from a
predetermined direction in a state where the surface plasmon resonance sensor chip
is held by said holding means (211);
light receiving means (213) for receiving the light reflected from the sensor surface;
measuring means (213) for measuring the intensity of the light reflected by each said
diffraction grating surface (251-254) and received by said light receiving means (213);
determining means (214) for determining, for each of the reaction area and the non-reaction
area, the variation between the intensity, measured by said measuring means (213),
of the reflected light due to each said diffraction grating surface (251-254) and
the intensity of the light reflected when any sample is not in contact with the sensor
surface; and
analyzing means (214) for selecting, for each of the reaction area and the non-reaction
area, a diffraction grating surface (251-254) whose determined variation of the reflected-light
intensity is within a predetermined allowable range for determination, and for quantitatively
and/or qualitatively analyzing the sample based on the variation of the reflected-light
intensity of the selected reaction area and the variation of the reflected-light intensity
of the selected non-reaction area.
66. An apparatus for quantitatively and/or qualitatively analyzing a sample using a surface
plasmon resonance sensor chip as defined in claim 43, comprising:
holding means (211) for holding the surface plasmon resonance sensor chip;
sample introducing means (282) for assigning a plurality of different samples to said
plural flow channels (280), respectively, and for introducing each of the plural samples
into the respective flow channel (280) in a state where the surface plasmon resonance
sensor chip is held by said holding means (211);
light irradiating means (212) for irradiating the sensor surface with light from a
predetermined direction in a state where each sample is introduced into the respective
flow channel (280) by said sample introducing means (282);
light receiving means (213) for receiving the light reflected from the sensor surface;
measuring means (213) for measuring the intensity of the light reflected by each said
diffraction grating surface (251-254) and received by said light receiving means (213);
determining means (214) for determining the variation between the intensity, measured
by said measuring means (213), of the reflected light due to each said diffraction
grating surface (251-254) and the intensity of the light reflected when any sample
does not flow through each said flow channel (280); and
analyzing means for selecting, for each said flow channel (280), a diffraction grating
surface (251-254) whose variation, determined by said determining means (214), of
the reflected-light intensity is within a predetermined allowable range for determination,
and for quantitatively and/or qualitatively analyzing each sample flowing through
the respective flow channel (280) based on the variation of the reflected-light intensity
of the diffraction grating surface (251-254) selected for each said flow channel (280).
67. An apparatus for quantitatively and/or qualitatively analyzing a sample using a surface
plasmon resonance sensor chip as defined in claim 44, comprising:
holding means (211) for holding the surface plasmon resonance sensor chip;
sample introducing means (282) for assigning a plurality of different samples to said
plural flow channels (280), respectively, and for introducing each of the plural samples
into the respective flow channel (280) in a state where the surface plasmon resonance
sensor chip is held by said holding means (211);
light irradiating means (212) for irradiating the sensor surface with light from a
predetermined direction in a state where each sample is introduced into the respective
flow channel (280) by said sample introducing means (282);
light receiving means (213) for receiving the light reflected from the sensor surface;
measuring means (213) for measuring the intensity of the light reflected by each said
diffraction grating surface (251-254) and received by said light receiving means (213);
determining means (214) for determining, for each of the reaction area and the non-reaction
area, the variation between the intensity, measured by said measuring means (213),
of the reflected light due to each said diffraction grating surface (251-254) and
the intensity of the light reflected when any sample does not flow through each said
flow channel (213); and
analyzing means (214) for selecting, for each said flow channel (280) and for each
of the reaction area and the non-reaction area, a diffraction grating surface (251-254)
whose variation, determined by said determining means (214), of the reflected-light
intensity is within a predetermined allowable range for determination, and for quantitatively
and/or qualitatively analyzing each sample flowing through the respective flow channel
(280), based on the variation of the reflected-light intensity of the selected reaction
area and the variation of the reflected-light intensity of the selected non-reaction
area for each said flow channel (280).
68. An apparatus as defined in one of claims 58-67, wherein it further comprises sample
separating means (292) for separating the sample by physical and/or chemical action
prior to introducing the sample to the surface plasmon resonance sensor chip.
69. An apparatus as defined in claim 68, wherein said sample separating means (292) is
operable to separate the sample by a separation technique using at least one of liquid
chromatography, HPLC (high performance liquid chromatography), capillary electrophoresis,
microchip electrophoresis, flow injection, and microchannel.
70. An apparatus as defined in one of claims 58-69, wherein
the target species is a light-emitting substance,
said light receiving means (213) is operable to detect the light emitted from the
light-emitting substance that is bound to the binding substance, and
said analyzing means (214) is operable to quantitatively and/or qualitatively analyze
the sample with consideration given to the detection result of the light emission
by said light receiving means (213).
71. A surface plasmon resonance sensor chip comprising:
a metal layer (23) along whose surface a surface plasmon wave can be induced by light
irradiation; and
a diffraction grating curved surface (25) disposed in the vicinity of said metal layer
(23), said diffraction grating curved surface (25) having a diffraction grating with
a uniform groove orientation and a uniform groove pitch so as to generate an evanescent
wave upon light irradiation;
wherein said diffraction grating curved surface (25) has a curved surface form
in a convex shape whose light-irradiated side bulges out, and is disposed so as to
be perpendicular to a specific plane (S1), which is perpendicular to a predetermined
reference plane (S0), and the diffraction grating is formed in such a manner that
the groove orientation is perpendicular to the specific plane (S1).
72. A surface plasmon resonance sensor chip comprising:
a metal layer (233) and a diffraction grating (235) formed in the vicinity of a sensor
surface, which comes in contact with a sample; and
a resonance area (238a-238d), formed on the sensor surface (231a), for causing a resonance
phenomenon of a surface plasmon wave, which is induced along the surface of said metal
layer (233), and an evanescent wave, which is generated by the action of the diffraction
grating, upon light irradiation;
wherein said resonance area (238a-238d) has a plurality of continuous areas (238a-238d)
discretely formed on the sensor surface (231a), and at least one continuous area (238a-238d)
among the plural continuous areas (238a-238d) has a diffraction grating whose at least
one of the groove pitch and the groove orientation is different from those of the
remaining continuous areas (238a-238d).
73. A surface plasmon resonance sensor chip comprising:
a metal layer (233) and a diffraction grating (235) formed in the vicinity of a sensor
surface, which comes in contact with a sample; and
a resonance area , formed on the sensor surface, for causing a resonance phenomenon
of a surface plasmon wave, which is induced along the surface of said metal layer
(233), and an evanescent wave, which is generated by the action of the diffraction
grating, upon light irradiation;
wherein said resonance area is formed continuously on the sensor surface, and
the groove orientations of the diffraction grating (225) are uniform while the groove
pitches of the diffraction grating (225) have a continuous or discontinuous distribution.